Antimicrobial activity of gemfibrozil and related compounds and derivatives and metabolites thereof

ABSTRACT

The present invention provides a method of selecting a compound which inhibits the enzymatic activity of enoyl reductase which comprises: (A) contacting enoyl reductase with the compound linked to an acyl carrier protein; (B) measuring the enzymatic activity of the enoyl reductase of step (A) compared with the enzymatic activity of enoyl reductase in the absence of the compound and selecting the compound which inhibits the enzymatic activity of enoyl reductase.

This application is a continuation and claims priority of U.S. Ser. No.09/438,144, filed Nov. 10, 1999, now U.S. Pat. No. 6,531,291, issuedMar. 11, 2003, the contents of which are incorporated herein byreference.

The invention disclosed herein was made with Government support underGrant Nos. AI23549 and AI20516 from NIAID. Accordingly, the U.S.Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced by anumber in brackets. Full citations for these publications may be foundlisted by number at the end of the specification immediately precedingthe claims. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art as known to those skilledtherein as of the date of the invention described and claimed herein.

Gemfibrozil (GFZ) is a compound that has been utilized as a drug forincreasing intracellular accumulation of hydrophilic anionic agents(U.S. Pat. No. 5,422,372, issued Jun. 6, 1995) and as a lipid regulatingcomposition (U.S. Pat. No. 4,859,703, issued Aug. 22, 1989). Gemfibrozilhas been shown to be effective in increasing the amount of cholesterolexcreted in to bile. (Ottmar Leiss et al., Metabolism, 34(1):74-82(1985)). Gemfibrozil is described in U.S. Pat. No. 3,674,836 and in TheMerck Index, 11 ed., Merck & Co., Inc. Rahway, N.J. 1989; #4280.Gemfibrozil, a drug which therapeutically lowers triglycerides andraises HDL-cholesterol levels, previously has not been reported to haveantimicrobial activity. (Brown, 1987; Oliver et al., 1978 and Palmer etal., 1978).

SUMMARY OF THE INVENTION

The present invention provides for a method of inhibiting activity of anenoyl reductase enzyme in a cell which comprises contacting the cellwith a compound having the structure:

-   wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ is independently selected    from the group consisting of: —H, —F, —Cl, —Br, —I, —OH, —OR₇, —CN,    —COR₇, —SR₇, —N(R₇)₂, —NR₇—COR₈, —NO₂, —(CH₂)_(p)—OR₇, —COSR₇,    —COOH, —CONH₂, —NH₂, a straight chain or branched, substituted or    unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀    cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl,    acyl, phenyl, substituted phenyl, or heteroaryl;-   wherein L is alternatively —N—, —S—, —O— or —C—;-   wherein R₇ is independently selected from the group consisting of    —H, —F, —Cl, —Br, —I, —OH, —CN, —COH, —SH₂, —NH₂, —NHCOH,    —(CH₂)_(p)OH, a straight chain or branched, substituted or    unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀    cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl,    acyl, phenyl, substituted phenyl, or heteroaryl;-   wherein A is selected from the group consisting of —N₂—, —NH—,    —C═C═CH₂—, —C≡C—C₂HOH—, —C≡C—CH₂—, —CH₂—CH₂—O—, —CH₂—CH₂—CH₂—O—,    —S—, —S (═O)₂—, —C═O—, —C═O—O—, —NH—C═O—, —C═O—NH—;-   wherein Q is independently an integer from 1 to 10, or if Q is 1, A    may be a (C₁-C₁₀)-alkyl chain, (C₁-C₁)—alkenyl chain or    (C₁-C₁₀)-alkynyl chain which is branched or unbranched, substituted    or unsubstituted and is optionally interrupted 1 to 3 times by —O—    or —S— or —N—;-   wherein X is —CO₂—, —CH═CH₃, phenyl, substituted phenyl, or    heteroaryl, —O-phenyl(CH₃)₂, —C(CH₂)₂—CO—NH₂, —C(CH₂)₂—COOH;    or a pharmaceutically acceptable salt or ester thereof, which    compound is present in a concentration effective to inhibit activity    of the enzyme.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Comparison of fatty acids synthesized by E. coli [5], and L.pneumophila [6, 7]. The number immediately preceding the colon refers tothe number of carbons, the number following the colon refers to thenumber of double bonds, the superscript number refers to the location ofthe double bonds. a=anteiso; i=iso; OH=hydroxy.

FIG. 2. Fatty acid synthesis in E. coli. Initiation of fatty acidsynthesis occurs with the condensation of acetyl-CoA with malonyl-ACP,or, conversion of acetyl-CoA to acetyl-ACP prior to condensation withmalonyl-ACP. Subsequent elongation occurs though the sequential additionof two carbon units using malonyl-ACP as the donor. Elongation occursthrough a four step process in which the first step, condensation, ismediated by β-ketoacylsynthase (FabB, FabF, FabH); the second step,reduction, is mediated by β-ketoacyl reductase (FabG); the third step,dehydration, is mediated through β-hydroxyacyl dehydratase (Fab Z); andthe fourth and final step, reduction, is mediated through enoylreductase (FabI). Unsaturated fatty acids are synthesized by thediversion of β-hydroxydecanol-ACP to FabA which catalyzes the formationof a double bond and then returns the unsaturated fatty acid to thecycle. The double lines indicate points where compounds act to inhibitfatty acid synthesis. The compounds are: DZB, diazoborines; ETH,ethionamide; INH, isoniazid; CER, cerulenin; TLM, thiolactomycin; NAG,3-decenoyl-N-acetylcysteamine.

FIG. 3. GFZ inhibits intracellular multiplication of L. pneumophila inphorbol myristate acetate-differentiated HL-60 cells. HL-60 cells weredifferentiated into macrophages by treatment with PMA for 48 hours,plated as a monolayer in the wells of a microtiter plate, andsynchronously infected with L. pneumophila (final multiplicity ofinfection of 0.01). After 2 hrs, the cells were washed to removeextracellular bacteria, overlaid with fresh RPMI-2 mM GLN-10% NHSwithout or with GFZ (100 μg/ml), and incubated at 37° C. At the timesindicated, the cells and medium were harvested and assayed for L.pneumophila. Illustrated is an experiment typical of three performed.Each point is the average of three separate wells (+/−) the SEM.

FIG. 4. GFZ protects HL-60 cells from the cytotoxic effects of an L.pneumophila infection over a five day incubation period. HL-60 cellswere differentiated and infected in microtiter wells as described inMaterials and Methods. GFZ was added to a final concentration of 0 or100 μg/ml 2.5 hours post-infection. After 5 days, the dye MTT was added,and the A₅₇₀ of each well was measured. The A₅₇₀ value is proportionalto the number of viable macrophages in the wells. Each point is theaverage of six separate wells (+/−) the SEM. This experiment isrepresentative of three experiments, all of which yeilded similarresults.

FIG. 5. GFZ inhibits the first round of intracellular multiplication ofL. pneumophila in PMA-differentiated HL-60 cells. PMA-differentiatedHL-60 cells at a concentration of 4×10⁶ cells/ml were synchronouslyinfected with L. pneumophila at an MOI of 0.01 in suspension. 100 alaliquots were plated in the wells of a 96 well microtiter plate. Theplates were centrifuged to pellet the cells and bacteria at the bottomof the wells. The monocytes were allowed to internalize the bacteria for2.5 hours at 37° C. prior to incubating with gentamicin 100 μg/ml for0.5 hours 37° C. The gentamicin-containing medium was then washed away,and replaced with fresh medium without or with GFZ 100 μg/ml.Intracellular multiplication was measured by lysing the monolayers, andtitering the combined lysate and supernatant at the times indicated.Each point is the average (+/−) the SEM of three separate cultures. Thisexperiment is representative of three such experiments.

FIG. 6. GFZ inhibits L. pneumophila growth within human monocyte-derivedmacrophages. Human blood monocytes, maintained in culture for five days,were suspended in fresh medium and infected with L. pneumophila at a MOIof 0.01. 100 μl of the infection mixture was aliquoted to the wells of a96 well plate, pelleted at 220 g and 880 g to pellet the bacteria andmacrophages, and incubated for 2.5 hr at 37° C. to allow the cells tointernalize the bacteria. 100 μl of fresh medium containing 2× the finaldrug concentration was then added to each well, and incubated at 37° C.At the indicated times, the cells and medium were harvested and assayedfor L. pneumophila CFUs. GFZ=gemfibrozil; BZF=bezafibrate;CFA=clofibrate. Each point represents the average of 3 wells (+/−) theSEM. This experiment is representative of three.

FIG. 7. Structures of the fibric acids used-in these experiments;gemfibrozil (GFZ), clofibrate (CFX), bezafibrate (BZF), fenofibrate(FNF). Note that GFZ, CFA, BZF, were used as the free acids, while FNFwas used as the isopropyl ester.

FIG. 8. Bacteria were screened for sensitivity to gemfibrozil using azone of inhibition assay. The assay was performed by overlaying bacteriaon a suitable nutrient agar plate, adding a disk containing 2 mggemfibrozil to the plate, and incubating the plate at the appropriatetemperature until bacterial growth was apparent. The presence of-a zoneof inhibition was considered positive for sensitivity. The fraction inparenthesis after each bacterial species indicates how many of thestrains tested of each species was sensitive to GFZ.

FIG. 9. GFZ zones of inhibition for S. aureus and S. epidermidis on LBversus TSB agar medium. Four ATTC S. aureus strains and one S.epidermidis strain were grown overnight in Brain-Heart Infusion (BHI)broth. The cultures were diluted to 10⁷ and 10⁶ CFUs/ml in BHI, and 100μl aliquots of each dilution were plated on LB or TSB agar in duplicate.The plates were incubated at 37° C. overnight prior to measuring thediameter of the zones in mm.

FIG. 10. Susceptibility of M. tuberculosis to GFZ. Twenty seven M.tuberculosis strains, demonstrating different drug resistance profiles,were tested for sensitivity to gemfibrozil. OADC-enriched Middlebrookagar plates with quadrants containing 0, 100, or 200 μg/ml of GFZ inwere prepared. 100 μls of a standard dilution of each resuspended M.tuberculosis strain in sterile water was added to each quadrant, and theplates were incubated for three weeks at 37° C. No growth was indicatedby (O); quadrants containing fewer than 50 colonies were counted, andthe numbers given are the average number of colonies in duplicatequadrants ; quadrants containing 50-100 colonies are indicated by a (+);quadrants containing 100-200 colonies are indicated by a (++); quadrantscontaining 200-500 colonies are indicated by a (+++); and quadrants withconfluent growth are indicated by (++++). The drugs to which each strainare resistant are indicated to the left of each strain; S=streptomycin 2μg/ml; I=isoniazid 1 μg/ml; R=rifampin 1 μg/ml; E=ethambutol 5 μg/ml;K=kanamycin 6 μg/ml; O=ofloxacin 4 μg/ml; C=ciprofloxacin 2 μg/ml;R^(L)=low level resistance to isoniazid at 0.2 μg/μl, but sensitive toisoniazid at higher concentrations.

FIG. 11. GFZ inhibits the growth of M. tuberculosis strains in 7H9broth. Approximately 10⁷ bacteria were added to 5 mls of Middlebrook 7H9broth with glycerol and incubated at 37° C. After 21 days the cultureswere visually assessed for turbidity.

FIG. 12. F4b has increased resistance to GFZ in an HL-60 intracellularinfection assay. HL-60 cells were differentiated into macrophages withPMA, plated as a monolayer in the wells of a microtiter plate, andsynchronously infected with L. pneumophila F4b or L. pneumophilaPhiladelphia 1 (final multiplicity of infection of 0.01). After 2 hrs,the cells were washed to remove non-phagocytosed and non-adherentbacteria, overlaid with fresh RPMI-2 mM GLN-10% NHS without or with GFZ(100 μg/ml), and incubated at 37° C. in a humidified atmospherecontaining 95% air and 5% CO₂. At the times indicated, the cells andmedium were harvested and assayed for L. pneumophila. Illustrated is anexperiment typical of two performed. Each point is the average of threeseparate wells (+/−) the SEM.

FIG. 13. Structural analogs of GFZ tested for activity against wild typeL. pneumophila Phill and L. pneumophila F4b by a zone of inhibitionassay. Salicylate=SAL; 4-hydroxyphenylpropionic acid (4-HPA);3,4-hydroxyphenylpropionic acid (3,4-HPA); gemfibrozil (GFZ).

FIG. 14. Structural analogs of GFZ demonstrate antibacterial activityagainst L. pneumophila (P1) and the L. pneumophila derived mutant F4b.Sterile disks containing either 2.5 mg or 1.0 mg of GFZ,3(p⁴-hydroxyphenyl)propionic acid (4-HPA), 3,4-dihydroxyphenyl propionicacid (3,4-HPA), and 2-hydroxybenzoic acid or salicylate (SAL) were addedto BCYE plates overlaid with L. pneumophila Philadelphia 1 or F4b andincubated at 37° C. After four days the diameters of the zones weremeasured.

FIGS. 15A-D. Effect of GFZ on the accumulation of electron-lucentinclusions by L. pneumophila Philadelphia 1. (A and B) Electronmicrographs of L. pneumophila grown for three days on CYE agar at 37° C.without (10,000×) (A) or with (20,000×) (B) GFZ (30 μg/ml). (C and D)Fluorescence micrographs (100×) of Nile Blue A stained L. pneumophilagrown for three days on CYE agar at 37° C. without (C) or with (D) GFZ(30 μg/ml).

FIG. 16. Effect of GFZ on 3-HB content of L. pneumophila. L. pneumophilawere grown on CYE agar with GFZ (30 μg/ml) or without GFZ for 3 days at37° C., harvested and lyophilized. 0.16 mg of benzoic acid (internalstandard) was added to 40 mg of each lyophilized sample, and thecorresponding propyl esters were formed by hydrochloric acidpropanolysis. The amount of 3-HB in each L. pneumophila sample wascalculated from a standard curve generated by derivitizing known amountsof 3-HB and benzoic acid to their corresponding propyl esters. Both thestandard curve and the L. pneumophila analyses were performed intriplicate using three sets of independently prepared samples for each.

FIG. 17. Putative interactions between the fatty acid synthesis pathwayand the PHB synthesis pathway. There are four reactions in each cycle offatty acid elongation. The first is condensation of malonyl-ACP withacetyl-ACP or a longer chain acyl-ACP to form a ketoester. In E. coli,this reaction can be catalyzed by FabH, FabB, or FabF. In organisms thatsynthesize branched chain fatty acids such as Legionella sp.,butyryl-ACP, rather than acetyl-ACP, is often preferred for condensationwith malonyl-ACP for initiation of fatty acid synthesis. The second stepinvolves the NADPH-dependent reduction of the ketoester catalyzed byFabG. The third step involves dehydration of the substrate by FabA orFabZ to form enoyl-ACP. Reduction of the enoyl-ACP by FabI, an enoylreductase, produces an acyl-ACP that can be further elongated byadditional cycles of fatty acid synthesis or utilized by the cell forphospholipid synthesis. Due to the instability of the enoyl-ACPcompound, inhibition of FabI has been shown to result in theaccumulation β-hydroxybutyryl-ACP in E. coli. In L. pneumophila,inhibition of fatty acid synthesis could result in accumulation ofacetyl-CoA or butyryl-CoA. Both are primers for fatty acid synthesis inbacteria, like L. pneumophila, which synthesize branched chain fattyacids. Alternatively, PhaG from Psuedomonas putida is known to catalyzethe conversion of β-hydroxybutyryl-ACP into β-hydroxybutyryl-CoA, asubstrate for PHB synthesis. L. pneumophila may contain a homolog ofthis P. putida enzyme.

FIG. 18. PHB synthesis pathway. PHB synthesis from acetyl-CoA generallyoccurs through a three step biosynthetic pathway. The first stepinvolves condensation by the β-ketothiolase, PhbA, of two acetyl CoAmolecules to form acetoacetyl-CoA. The second step involves theformation of 3-hydroxybutyryl-CoA (3-HB-CoA) by the acetoacetyl-CoAdehydrogenase, PhbB. The final step involves polymerization of the3-HB-CoA monomers by the PHB synthase, PhbC. However, certain species ofbacteria have the ability to utilize substrates from other metabolicpathways such as fatty acid synthesis and β-oxidation to form PHA. Forexample, PhaG of Pseudomonas putida, mediates the conversion of thefatty acid synthesis intermediate 3-HB-ACP to (R)-3-HB-CoA [110], whilePhaJ, identified in Aeromonas caviaes, mediates the hydration ofenoyl-CoA substrates from the β-oxidation pathway [111]. Completion ofβ-oxidation results in the formation of acetyl-CoA which can be utilizedas a substrate for either fatty acid synthesis or PHB synthesis.Accumulation of acetyl CoA, due to inhibition of either thetricarboxylic acid cycle or fatty acid synthesis, could result inincreased incorporation of acetyl-CoA into PHB. The lightly shadedcircles represent cycles which either synthesize or utilize substrates,represented by the dark shaded boxes, putatively involved in PHBsynthesis in L. pneumophila.

FIG. 19. PHB synthesis pathway. PHB synthesis from acetyl-CoA generallyoccurs through a three step biosynthetic pathway. The first stepinvolves the condensation of two acetyl CoA molecules to formacetoacetyl-CoA by the β-ketothiolase, PhbA. The second step involvesthe formation of 3-hydroxybutyryl-CoA (3-HB-CoA) by the acetoacetyl-CoAdehydrogenase, PhbB. The final step involves the polymerization of themonomer unit 3-HB-CoA by PHB synthase, PhbC. However, certain species ofbacteria have the ability to utilize substrates from other metabolicpathways to form PHB. For example, PhaG of Pseudomonas putida, mediatesthe conversion of the fatty acid synthesis intermediate 3-HB-ACP to(R)-3-HB-CoA [110], while Alcaligenes eutrophus has been demonstrated toutilize exogenous butyrate, presumably following its derivitization tobutyryl-CoA, oxidation to crotonyl-CoA, and hydration to (R)-3-HB-CoA[109]. PhaJ, identified in Aeromonas caviaes, mediates the hydration ofenoyl-CoA substrates from the β-oxidation pathway [111]. The doublelines indicate pathways, which when blocked, may increase theutilization of these intermediates by the PHB synthesis pathway.

FIG. 20. Fatty acid biosynthesis in bacteria. Precursors such asacetyl-CoA, butyryl-CoA, isobutyryl-CoA, valeryl-CoA and2-methylbutyryl-CoA are utilized by various bacterial species forcondensation with malonyl-CoA to initiate fatty acid synthesis.Subsequent elongation occurs though the sequential addition of twocarbon units using malonyl-CoA as the donor. Elongation occurs through afour step process in which the first step, condensation is mediated byβ-ketoacyl synthase; the second step, reduction is mediated byβ-ketoacyl reductase; the third step, dehydration, is mediated throughβ-hydroxyacyl dehydratase; and the fourth and final step, reduction, ismediated through enoyl reductase. Additional enzymes mediating reactionsallowing for fatty acyl modifications include β-hydroxydecanoldehydrase, which catalyzes double bond formation, and cyclopropyl fattyacid synthase, which catalyzes the formation of cyclopropane fattyacids. Regulation of fatty acid composition is achieved by mechanismsincluding multiple isoforms of enzymes with different substratespecificities and regulatory characteristics }[5], temperaturesensitivity }[10], and transcriptional control }[11].

FIG. 21. GFZ inhibits ¹⁴C-acetate incorporation into lipids in L.pneumophila. Increasing concentrations of GFZ (10, 25, 50, or 100 μg/ml)were added to L. pneumophila Philadelphia 1 cultures in the presence of¹⁴C-acetate. At the indicated times, 100 μl aliquots were TCAprecipitated and analyzed for ¹⁴C-acetate content by scintillationspectrometry. The experiment was repeated three times and the resultsshown here are of one experiment, representative of the three performed.

FIG. 22. F4b, a partially GFZ-resistant L. pneumophila variant displaysincreased resistance to GFZ-mediated inhibition of ¹⁴C-acetateincorporation into lipids. GFZ at the indicated concentrations (25, 50,75, 100 μg/ml) was added to a L. pneumophila F4b in AYE mediumcontaining ¹⁴C-acetate (5 μCi/ml). The bacteria were incubated at 37° C.for the indicated times. 100 μl aliquots were TCA precipitated andanalyzed for ¹⁴C content by scintillation spectrometry. 50% inhibitionof ¹⁴C-acetate incorporation relative to the control, was achieved at aGFZ concentration of 100 μg/ml (0.4 mM). This experiment was performedonly once. It is consistent with the finding that F4b has an MIC valueapproximately five times higher than that of wild type L. pneumophila.

FIG. 23. GFZ decreases ¹⁴C-acetate incorporation intochloroform/methanol extracts of L. pneumophila lipids. GFZ (10-100μg/ml) was added to L. pneumophila cultures in the presence of¹⁴C-acetate. After 60 min incubation at 37° C., the bacteria werepelleted and extracted with chloroform/methanol/double distilled H₂O(1:1:0.9). The extracts were assayed by scintillation spectrometry andautoradiography of TLC plates. By autoradiography, ¹⁴C-acetateincorporation into the various lipid species appeared equally inhibitedat every GFZ concentration tested. Lane 1: GFZ 0 μg/ml; Lane 2 GFZ 10μg/ml; Lane 3 GFZ 25 μg/ml; Lane 4 GFZ 50 μg/ml; Lane 5 GFZ 100 μg/ml.Liquid scintillation counting of duplicate samples indicated that 50%inhibition, relative to the control, was achieved at a GFZ concentrationof 10 μg/ml, or 40 uM. This experiment was performed only once. It isconsistent with the TCA precipitation results reported earlier in FIG.21.

FIG. 24. Comparison of TCA precipitation and chloroform/methanolextraction as a measure of GFZ inhibition of ¹⁴C-acetate incorporationin L. pneumophila lysates. GFZ or cerulenin at 0.4 mM or 2.0 mMconcentrations was added to L. pneumophila lysates in the presence of¹⁴C-acetate (5 μCi/ml), total volume 500 μl, and incubated at 37° C. forone hour. 200 μl of each incubation mixture was TCA precipitated, and300 μl was extracted with chloroforrn/methanol/double distilled H₂O(1:1:0.9). The amount of ¹⁴C-acetate incorporated into TCA precipitableor chloroform/methanol extractable material in each incubation mixturewas measured by scintillation counting and is expressed as a percent ofthe uninhibited control. This experiment was performed once. It isconsistent with the data from while cells (FIG. 21).

FIG. 25. Comparison of the effects of GFZ, bezafibrate, and cerulenin on¹⁴C-acetate incorporation in L. pneumophila lysates. GFZ, cerulenin(CER), or bezafibrate (BZF) at 2 mM, or EDTA at 10 mM, was added to L.pneumophila lysates in the presence of ¹⁴C-acetate (10 μCi/ml) intriplicate and incubated at 37° C. in a final reaction volume of 100 μl.One lysate was heated in a boiling water bath for ten minutes prior tothe addition of ¹⁴C-acetate. At 10, 20, and 30 minutes, the reactionswere TCA precipitated and pelleted. The TCA precipitate was thenextracted with chloroform/methanol/double distilled H₂O (1:1:0.9). Thechloroform/methanol extracts were assayed by scintillation spectrometryto determine relative rates of ¹⁴C-acetate incorporation into lipids.The experiment was repeated twice with similar results. The data shownare from a representative experiment.

FIG. 26. Structures of the GFZ analogs tested for inhibition of¹⁴C-acetate incorporation into L. pneumophila whole cells or lysates.SAL=salicylate; PAS=para-aminosalicylate; ASAL=acetylsalicylate;INH=isoniazid; CFA=clofibrate; 4-HPA=4-hydroxypropionate;3,4-HPA=3,4-hydroxypropionate; GFZ=gemfibrozil; TEVA (B-H)=TEVA analogs.

FIGS. 27A-B. Ability of GFZ analogs to inhibit ¹⁴C-acetate incorporationinto the TCA precipitates of intact L. pneumophila. TEVA analogs (A-H)were added to L. pneumophila in AYE medium containing ¹⁴C-acetate at aconcentration of 0.4 mM. At various time points 100 μl aliquots of thecultures were TCA precipitated and analyzed for ¹⁴C-acetate content byscintillation spectroscopy.

FIGS. 28A-B. TEVA analogs C and D inhibit fatty acid synthesis in L.pneumophila cultures. Increasing concentrations of (FIG. 28A) analog Cand (FIG. 28B) analog D were added to a L. pneumophila Philadelphia 1cultures in the presence of ¹⁴C-acetate. At the indicated time points,100 μl aliquots were TCA precipitated and the precipitates were analyzedfor ¹⁴C-acetate content by scintillation spectrometry. 50% inhibition,relative to the control, was achieved at a concentration of 40 μM. Datapresented are for one experiment using triplicate samples.

Ability of commercially available GFZ analogs to inhibit ¹⁴C-acetateincorporation into the TCA precipitates of intact L. pneumophila.Analogs were added to L. pneumophila in AYE medium containing¹⁴C-acetate at a concentration of 0.5 mM. At various time points 100 μlaliquots of the cultures were TCA precipitated and analyzed for¹⁴C-acetate content by scintillation spectroscopy.

FIG. 29. Sites of drug-mediated inhibition of fatty acid synthesis in E.coli. Initiation of fatty acid synthesis occurs with the condensation ofacetyl-CoA with malonyl-ACP, or, conversion of acetyl-CoA to acetyl-ACPprior to condensation with malonyl-ACP. Subsequent elongation occursthough the sequential addition of two carbon units using malonyl-ACP asthe donor. Elongation occurs through a four step process in which thefirst step, condensation, is mediated by β-ketoacyl syntheses (FabB,FabF, FabH); the second step, reduction is mediated by β-ketoacylreductase (FabG); the third step, dehydration, is mediated byβ-hydroxyacyl dehydratase (Fab Z); and the fourth and final step,reduction, is mediated by enoyl reductase. (FabI). Unsaturated fattyacids are synthesized by the diversion of β-hydroxydecanol-ACP to FabAwhich catalyzes the formation of a double bond and then returns theunsaturated fatty acid to the cycle. The double lines indicate pointswhere compounds act to inhibit fatty acid synthesis. The compounds thatinhibit at each site are; DZB, diazoborines; ETH, ethionamide; INH,isoniazid; TCN, triclosan; CER, cerulenin; TLM, thiolactomycin; NAG,3-decenoyl-N-acetylcysteamine.

FIG. 30. sequence alignment of enoyl reductases from L. pneumophiia E.coil, S. typhimuriurn, H. influenza, and M. tuberculosis (SEQ IDNOS:1-5). Completely conserved residues are indicated by bold type,while highly conserved residues the indicated by program an asterisk.The figure was produced using the clustal W program [143].

FIG. 31. Effect-of Temperature and GFZ on growth of E. Coli transformedwith an L. Pneumophila enoyl reductase homolog.

FIG. 32. Effect of oleate and palmitate supplementation on the growth ofE. coli FT100 and FT101 strains at 30° C. versus 43° C., without or withGFZ 500 μg/ml. An isogenic pair of E. coli strains, FT100 and FT101,transformed with vector-only (pCR2.1), or vector containing the L.pneumophila enoyl reductase gene (pCK1), were grown at 30° C. in LB(Km)(—NaCl) broth to late log/early stationary phase. Cultures were seriallydiluted, plated on low osmolarity (0.2% NaCl) LB(Km) plates without (−)or with (+) GFZ 500 μg/ml, and without or with 0.3 mM palmitate (PA) oroleate (OA) alone or in combination. The plates were incubated for 2days at 30° C. or 43° C., and the colonies counted. Solid bars=control;shaded bars=GFZ 500 μg/ml.

FIG. 33. Alignment of the FabX and FabT enoyl reductases from L.pneumophila (SEQ ID NOS:6 and 7).

FIG. 34. SDS-PAGE analysis of His-tagged L. pneumophila FabX and FabT.400 ml cultures of E. coli BL21 DE3 transformed with the pET15b:FabX,pET15b:FabT, or pET15b:FabI vectors were grown in LB-Amp broth at 37° C.to an OD of 1.0 at 600 nm. IPTG was added to a final concentration of 1mM and the cultures were incubated for an additional three hours.Bacterial pellets were harvested by centrifugation and lysed bysonication. The His-tagged proteins were purified by nickel columnchromatography. The purified products were eluted from the column with400 mM imidazole. Aliquots were analyzed for purity by SDS-PAGE.1=protein standard; 2=L. pneumophila FabX lysate; 3=flow through afterwashing the FabX column with 40 mM imidazole; 4=400 mM imidazole eluateof the FabX column; 5=L. pneumophila FabT lysate; 6=flow through afterwashing the FabT column with 40 mM imidazole; 7=400 mM imidazole eluateof the FabT column; 8=E. coli FabI lysate; 9=flow through after washingthe FabI column with 40 mM imidazole; 10=400 mM imidazole eluate of theFabI column.

FIG. 35. Comparison of the specific activities of L. pneumophila FabTand FabX, and of E. coli FabI, for crotonoyl-CoA. His-tagged FabX, FabT,or FabI, was incubated with increasing concentrations of crotonoyl-CoA(CCA) in the presence of excess NADH. The rate of NADH hydrolysis wasassessed for each enzyme for each concentration of CCA by measuring thedecrease in absorbance over time at A₃₄₀nm. Specific activities werecalculated. The specific activities for FabX and FabT are the averagefor two experiments (+/−) the SEM. The specific activity for was onlymeasured once.

FIG. 36. Structures of the enoyl reductase substrates and inhibitors.

FIGS. 37A-C. GFZ-CoA is a competitive inhibitor of enoyl reductaseactivity for L. pneumophila Fab X and FabT, and M. tuberculosis InhAusing dodecenoyl-CoA (DCA) as a substrate. Panel a=FabX; panel b=FabT;panel c=InhA. Reaction mixtures containing 100 mM NaPO₄ pH 7.4, 100 μMNADH, and enoyl reductase, were combined with varying concentrations ofDCA and GFZ. The ability of GFZ-COA to inhibit the enoyl reductaseactivity was assessed by measuring the change in absorbance over time atA₃₄₀nm as NADH was oxidized to NAD⁺. Concentrations of GFZ-CoA (μM)utilized are indicated in bold type next to the corresponding plot.Inhibition was competitive with regard to the DCA substrate.

FIG. 38. Flow chart of the methods utilized for the ³H-GFZ metaboliclabeling studies.

FIG. 39. 280 nm absorbance tracing for the T=120 minute >10 kDA FPLCsample. 500 ml of the <10 kDa filtrate was loaded onto a Superose™ FPLCcolumn and run at 0.5 ml/min with a paper speed of 0.5 mm/min. Eachsquare is 2 mm².

Therefore, the large boxes correspond to 5 mls or 10 minutes, while thesmall boxes correspond to 1 ml or 2 minutes. 0.5 ml fractions werecollected post 280 nm detection. The injection is on the right side ofthe tracing and the four major peaks are numbered in order ofappearance. The radioactive fractions are indicated as dots along theX-axis.

FIG. 40. FPLC analysis of cytoplasmic extracts of L. pneumophilaincubated with ³H-GFZ. L. pneumophila were incubated with ³H-GFZ in AYEbroth at 37° C. Samples were collected at the times indicated. Thesamples were pelleted, washed, lysed, and filtered through 30 and 10 kDaCentricon filters. The filtrate was applied to a Superose12™ FPLCcolumn. Fractions were collected and CPMs assessed by liquidscintillation spectroliletry.

Percentage of CPMs in each of the three major PLC peaks relative tototal CPMs for samples harvested at the time points indicated.

FIG. 41. HPLC of the ³H-GFZ standard. ³H-GFZ (lμCi) dissolved in 100%EtOH was applied to a reverse phase C18 μBondapack™ column. 0.5 mlfractions were collected every thirty seconds and assessed for CPMs byliquid scintillation spectrometry.

FIGS. 42. HPLC of the GFZ-CoA standard. 500 μl of a mixture of CoA (30%)and GFZ-CoA (70%) dissolved in water at a concentration of 500 μg/ml wasadded to a reverse phase C18 μBondaPack column. 5.0 ml fractions werecollected every thirty seconds and alyzed at OD=262 nm. Fractionseluting at 5-7 min (fractions #10-14) represent GFZ-CoA the fractioneluting at 4 min (fraction #8) represents CoA.

FIGS. 43A-D Summary of the HPLC data from FPLC fraction #53. Filteredlysates of L. pneumophila incubated at 37° C. with ³H-GFZ for theindicated length of time were applied to an FPLC column and fractionswere collected. Fraction #53 from the third peak to elute from this FPLCcolumn was applied to a reverse phase HPLC column. Fractions from theHPLC column were collected and assessed by liquid scintillationcounting. Fractions eluting from 12.5-15 minutes (fractions #25-30)contained radioactivity and co-chromatograph with ³H-GFZ as determinedearlier (FIG. 8-5). a) T=5 min; b) T=30 min c) T=60 min d) T=180 min.

FIGS. 44A-D. summary of the HPLC data from FPLC fraction #45. Filteredlysates of L. pneumophila incubated with ³H-GFZ for the indicated lengthof time were applied to an FPLC column and fractions were collected. #45from the second peak to elute from the FPLC column was applied to areverse phase HPLC column and fractions were collected and assessed byliquid scintillation spectrometry. Fractions collected from 3-4 minutes(fractions #6-8) contained radioactivity and co-chromatographed with thebreakdown product in the ³H-GFZ stock a determined earlier (FIG. 8-5).Fractions collected from 12.5-15 minutes (fractions #25-30) alsocontained radioactivity and co-chromatographed with the GFZ stock asdetermined earlier, a) T=5 min; b) T=30 min c) T=60 min d) T=180 min.

FIGS. 45A-D. Summary of the HPLC data from FPLC fraction #38. Filteredlysates of L. pneumophila incubated with ³H-GFZ for the indicated lengthof time were applied to an FPLC colum and fractions were collected.Fraction #38 from the first peak to elute was applied to a reverse phaseHPLC column and fractions were collected and assessed by liquidscintillation counting. Fractions collected from 3-4 minutes (fractions#6-8) contained radioactivity and co-chromatographed with thecontaminant in the ³H-GFZ preparation as determined earlier (FIG. 42).Fractions collected from 12.5-15 minutes (fractions #25-30) containedradioactivity and co-chromatographed with GFZ as determined earlier.Fractions collected from 6-8 minutes (fractions #12-16) containedradioactivity and co-chromatographed with GFZ-CoA as determined earlier,a) T=5 min; b) T=30 min c) T=60 min; d) T=180 min.

FIG. 46. Percentage of CMPs in the putative ³H-GFZ-CoA peak, realativeto total CPMs for each sample, increases as the incubation period of L.pneumophila with ³H-GFZ increases.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a method of inhibiting activity of anenoyl reductase enzyme in a cell which comprises contacting the cellwith a compound having the structure:

-   wherein each of R¹, R₂, R₃, R₄, R₅ and R₆ is independently selected    from the group consisting of: —H, —F, —Cl, —Br, —I, —OH, —OR₇, —CN,    —COR₇, —SR₇, —N(R₇)₂, —NR₇—COR₈, —NO₂, —(CH₂)_(p)—OR₇, —COSR₇,    —COOH, —CONH₂, —NH₂, a straight chain or branched, substituted or    unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀    cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl,    acyl, phenyl, substituted phenyl, or heteroaryl;-   wherein L is alternatively —N—, —S—, —O— or —C—;-   wherein R₇ is independently selected from the group consisting of    —H, —F, —Cl, —Br, —I, —OH, —CN, —COH, —SH₂, —NH₂, —NHCOH,    —(CH₂)_(p)OH, a straight chain or branched, substituted or    unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀    cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl,    acyl, phenyl, substituted phenyl, or heteroaryl;-   wherein A is selected from the group consisting of —N₂—, —NH—,    —C═C═CH₂—, —C≡C—C₂HOH—, —C≡C—CH₂—, —CH₂—CH₂—O—, —CH₂—CH₂—CH₂—O—,    —S—, —S(═O)₂—, —C═O—, —C═O—O—, —NH—C═O—, —C═O—NH—;-   wherein Q is independently an integer from 1 to 10, or if Q is 1, A    may be a (C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or    (C₁-C₁₀)-alkynyl chain which is branched or unbranched, substituted    or unsubstituted and is optionally interrupted 1 to 3 times by —O—    or —S— or —N—;-   wherein X is —CO₂—, —CH═CH₃, phenyl, substituted phenyl, or    heteroaryl, —O-phenyl(CH₃)₂, —C(CH₂)₂—CO—NH₂, —C(CH₂)₂—COOH;    or a pharmaceutically acceptable salt or ester thereof, which    compound is present in a concentration effective to inhibit activity    of the enzyme.

In one embodiment, A is selected from the group consisting of(C₁-C₁₀)-alkylene chain, (C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or(C₁-C₁₀)-alkynyl chain which is branched or unbranched, substituted orunsubstituted and is optionally interrupted 1 to 3 times by —O— or —S—or —N—.

In another embodiment of the present invention

-   -   R₁=R₄=CH₃ or —OH,    -   R₂=R₃=R₅=R₆ H or —OH,    -   A=CH₂,    -   and Q=3.

In another embodiment of the present invention,

-   -   R₃=Cl,    -   R₁=R₂=R₄=R₅=R₆=H or —OH,    -   and Q=0.

In another embodiment of the present invention,

-   -   R₆=CH(CH₃)₂,    -   R₁=R₂=R₄=R₅=H or —OH,    -   and Q=0.

In another embodiment of the present invention,

-   -   R₃=Cl,    -   R₆=C₂H₅,    -   R₁=R₂=R₄=R₅=H or —OH,    -   and Q=0.

In another embodiment, the enzyme is in a bacterium or the enzyme is inan eukaryotic cell. In one embodiment, the cell is a yeast cell, thecell is a fungus, the cell is a plant cell, or the cell is a protozoancell.

In one embodiment, the concentration of the compound or the metabolitethereof is from about 5 μg/ml to about 200 μg/ml. In a preferredembodiment, the concentration of the compound is 100 μg/ml. In anotherpreferred embodiment, the compound is administered at a concentration of150 micrograms/ml/kg body weight.

The present invention also provides for a method of selecting a compoundwhich inhibits the enzymatic activity of enoyl reductase whichcomprises: (A) contacting enoyl reductase with the compound or ametabolite thereof; (B) measuring the enzymatic activity of the enoylreductase of step (A) compared with the enzymatic activity of enoylreductase in the absence of the compound or the metabolite thereof,thereby selecting a compound or metabolite thereof which inhibits theenzymatic activity of enoyl reductase.

In one embodiment, the metabolite is a CoA metabolite. In anotherembodiment, the metabolite is an ACP metabolite. One of skill-in the artwould know of other metabolites which would be produced or generatedduring the fatty acid synthetic pathway.

The present invention provides a method of selecting a compound whichinhibits the enzymatic activity of enoyl reductase which comprises: (A)contacting enoyl reductase with the compound or metabolite thereof,wherein the compound or metabolite thereof contacts enoyl reductase atthe site at which gemfibrozil contacts enoyl reductase;(B) measuring theenzymatic activity of the enoyl reductase of step (A) compared with theenzymatic activity of enoyl reductase in the absence of the compound ormetabolite thereof, thereby selecting a compound which inhibits theenzymatic activity of enoyl reductase.

The present invention also provides for a method for inhibiting growthof a bacterium which consists essentially of contacting the bacteriumwith a compound having the structure:

-   wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ is independently selected    from the group consisting of: —H, —F, —Cl, —Br, —I, —OH, —OR₇, —CN,    —COR₇, —SR₇, —N(R₇)₂, —NR₇—COR₈, —NO₂, —(CH₂)_(p)—OR₇, —COSR₇,    —COOH, —CONH₂, —NH₂, a straight chain or branched, substituted or    unsubstituted C₁—C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀    cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl,    acyl, phenyl, substituted phenyl, or heteroaryl;-   wherein L is alternatively —N—, —S—, —O— or —C—;-   wherein R₇ is independently selected from the group consisting of    —H, —F, —Cl, —Br, —I, —OH, —CN, —COH, —SH₂, —NH₂, —NHCOH,    —(CH₂)_(p)OH, a straight chain or branched, substituted or    unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀    cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl,    acyl, phenyl, substituted phenyl, or heteroaryl;-   wherein A is selected from the group consisting of —N₂—, —NH—,    —C═C═CH₂—, —C≡C—C₂HOH—, —C≡C—CH₂—, —CH₂—CH₂—O—, —CH₂—CH₂—CH₂—O—,    —S—, —S(═O)₂—, —C═O—, —C═O—O—, —NH—C═O—, —C═O—NH—;-   wherein Q is independently an integer from 1 to 10, or if Q is 1, A    may be a (C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or    (C₁-C₁₀)-alkynyl chain which is branched or unbranched, substituted    or unsubstituted and is optionally interrupted 1 to 3 times by —O—    or —S— or —N—;-   wherein X is —CO₂—, —CH═CH₃, phenyl, substituted phenyl, or    heteroaryl, —O-phenyl(CH₃)₂, —C(CH₂)₂—CO—NH₂, —C(CH₂)₂—COOH;    or a pharmaceutically acceDtable salt or ester thereof, which    compound is present in a concentration effective to inhibit growth    of the bacterium.

In one embodiment, A is selected from the group consisting of(C₁-C₁)-alkylene chain, (C₁-C₁)-alkyl chain, (C₁-C₁₀)-alkenyl chain or(C₁-C₁₀)-alkynyl chain which is branched or unbranched, substituted orunsubstituted and is optionally interrupted 1 to 3 times by —O— or —S—or —N—.

In one embodiment of the present invention, the bacterium is Legionellapneumophila. In another embodiment, the bacterium is Nocardia sp. Inanother embodiment, the bacterium is Staph auereous. In anotherembodiment, the bacterium is Mycobacterium tuberculosis.

In one embodiment, the bacterium is in a eukaryotic cell.

In another embodiment, the concentration of the compound is from about 5μg/ml to about 100 μg/ml, or in a more preferred embodiment, theconcentration is from 20 μg/ml to 100 μg/ml.

The present invention also provides for a method for alleviating thesymptoms of a bacterial infection in a subject which comprisesadministering to the subject an amount of a compound having thestructure:

-   wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ is independently selected    from the group consisting of: —H, —F, —Cl, —Br, —I, —OH, —OR₇, —CN,    —COR₇, —SR₇, —N(R₇)₂, —NR₇—COR₈, —NO₂, —(CH₂)_(p)—OR₇, —COSR₇,    —COOH, —CONH₂, —NH₂, a straight chain or branched, substituted or    unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀    cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl,    acyl, phenyl, substituted phenyl, or heteroaryl;-   wherein L is alternatively —N—, —S—, —O— or —C—;-   wherein R₇ is independently selected from the group consisting of    —H, —F, —Cl, —Br, —I, —OH, —CN, —COH, —SH₂, —NH₂, —NHCOH,    —(CH₂)_(p)OH, a straight chain or branched, substituted or    unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀    cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl,    acyl, phenyl, substituted phenyl, or heteroaryl;-   wherein A is selected from the group consisting of —N₂—, —NH—,    —C═C═CH₂—, —C≡C—C₂HOH—, —C≡C—CH₂—, —CH₂—CH₂—O—, —CH₂—CH₂—CH₂—O—,    —S—, —S(═O)₂—, —C═O—, —C═O—O—, —NH—C═O—, —C═O—NH—;-   wherein Q is independently an integer from 1 to 10, or if Q is 1, A    may be a (C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or    (C₁-C₁₀)-alkynyl chain which is branched or unbranched, substituted    or unsubstituted and is optionally interrupted 1 to 3 times by —O—    or —S— or —N—;-   wherein X is —CO₂—, —CH═CH₃, phenyl, substituted phenyl, or    heteroaryl, —O-phenyl(CH₃)₂, —C(CH₂)₂—CO—NH₂, —C(CH₂)₂—COOH;    or a pharmaceutically acceptable salt or ester thereof, which    compound is present in a concentration effective to alleviate the    symptoms of a bacterial infection in the subject.

In one embodiment, the bacterial infection is associated with Legionellapneumophila, Mycobacterium tuberculosis, Bacillus subtilis, BacillusMegaterium, Rhodococcus sp., Staph epidermidis, Group A Streptococcussp., Coag neg Staphylococcus aureus or Nocardia sp.

In another embodiment, the bacterial infection is associated withLegionella pneumophila. In another embodiment, the bacterial infectionis associated with Mycobacterium tuberculosis.

In another embodiment, the effective concentration of thepharmaceutically acceptable compound is about 100 micrograms/ml.

In another embodiment, the subject is a human or an animal. In anotherembodiment, the bacterial infection is associated with Leprosy, Brucellaor Salmonella.

In another embodiment the concentration of the compound is from about 5μg/ml blood of the subject to about 200 μg/ml blood of the subject. Inanother embodiment the concentration of the compound is 100 μg/ml bloodof the subject. In another embodiment the administration to the subjectis oral, subcutaneous, intraveneous or intramuscular.

The present invention provides for a method of altering (inhibiting orenhancing) a biochemical pathway of fatty acid synthesis in a bacteriumwhich comprises contacting the bacterium with a compound having thestructure

-   wherein each of R₁, R₂, R₃, R₄, R₅ and R₆ is independently selected    from the group consisting of: —H, —F, —Cl, —Br, —I, —OH, —OR₇, —CN,    —COR₇, —SR₇, —N(R₇)₂, —NR₇—COR, —NO₂, —(CH₂)_(p)—OR₇, —COSR₇, —COOH,    —CONH₂, —NH₂, a straight chain or branched, substituted or    unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀    cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl,    acyl, phenyl, substituted phenyl, or heteroaryl;-   wherein L is alternatively —N—, —S—, —O— or —C—;-   wherein R₇ is independently selected from the group consisting of    —H, —F, —Cl, —Br, —I, —OH, —CN, —COH, —SH₂, —NH₂—NHCOH,    —(CH₂)_(p)OH, a straight chain or branched, substituted or    unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀    cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl,    acyl, phenyl, substituted phenyl, or heteroaryl;-   wherein A is selected from the group consisting of —N₂—, —NH—,    —C═C═CH₂—, —C≡C—C₂HOH—, —C≡C—CH₂—, CH₂—CH₂—O—, —CH₂—CH₂—CH₂—O—, —S—,    —S(═O)₂, —C═O—, —C═—O—, —NH—C═O—, —C═O—NH—;-   wherein Q is independently an integer from 1 to 10, or if Q is 1, A    may be a (C₁-C₁₀)-alkyl chain, (C₁-C₁₀)-alkenyl chain or    (C₁-C₁₀)-alkynyl chain which is branched or unbranched, substituted    or unsubstituted and is optionally interrupted 1 to 3 times by —O—    or —S— or —N—;-   wherein X is —CO₂—, —CH═CH₃, phenyl, substituted phenyl, or    heteroaryl, —O-phenyl(CH₃)₂, —C(CH₂)₂—CO—NH₂, —C(CH₂)₂—COOH;    or a pharmaceutically acceptable salt or ester thereof, which    compound is present in a concentration effective to alter the    pathway of fatty acid synthesis in the bacterium.

In one embodiment, the compound has the structure:

In another embodiment, the compound has the structure:

In another embodiment, the compound has the structure:

In another embodiment, the compound has the structure:

The present invention provides for a method of inhibiting activity of anenoyl reductase enzyme in a cell which comprises contacting the cellwith a compound having the structure:

-   wherein R₁ is selected from the group consisting of: —H, —F, —Cl,    —Br, —I, —OH, —OR₇, —CN, —COR₇, —SR₇, —N(R₇)₂, —NR₇—COR₈, —NO₂,    —(CH₂)_(p)—OR₇, —COSR₇, —COOH, —CONH₂, —NH₂, a straight chain or    branched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,    C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl,    methylene thioalkyl, acyl, phenyl, substituted phenyl, or    heteroaryl;-   wherein Q is independently an integer from 1 to 10;-   wherein R₇ is independently selected from the group consisting of    —H, —F, —Cl, —Br, —I, —OH, —CN, —COH, —SH₂, —NH₂, —NHCOH,    —(CH₂)_(p)OH, a straight chain or branched, substituted or    unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀    cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl, methylene thioalkyl,    acyl, phenyl, substituted phenyl, or heteroaryl;-   or a pharmaceutically acceptable salt or ester thereof, which    compound is present in a concentration effective to inhibit enoyl    reductase enzyme in the cell.

The method also includes use of a pharmaceutically acceptable salt orester thereof, which compound is present in a concentration effective toinhibit bacterial growth and thus alleviate the symptoms of thebacterial infection in the subject.

The present invention also provides for a pharmaceutical compositioncomprising a compound or metabolite thereof having any one of thestructures shown or described hereinabove and a pharmaceuticallyacceptable carrier.

The bacterial infection may be associated with a bacterium listed above.The subject may be a human or an animal. The bacterial infection may beassociated with Leprosy, Brucella or Salmonella. The concentration ofthe compound may be from about 5 μg/ml blood of the subject to about 200μg/ml blood of the subject. In one embodiment, the concentration of thecompound may be 100 μg/ml blood of the subject. The administration tothe subject may be oral.

As used herein Enoyl Reductase Enzyme includes enzymes having enoylreductase activity. Such enzymes may be bacterial enoyl reductases oreukaryotic enoyl reductases. Examples of bacterial enoyl reductasesinclude those from the bacterium listed above. The enoyl reductase maybe one of the enoyl reductases from L. Pneumophila. The enoyl reductasemay be a gene product of a gene that hybridizes with moderate or highstringency with the envM gene.

The enzyme may be in a bacterium. The bacterium may be Legionellapneumophila, Mycobacterium tuberculosis, Bacillus subtilis, BacillusMegadterium, Pseudomonas Oleovorans, Alcaligenes eutrophus, Rhodococcussp., Citrobacter freundi, Group A Streptococcus sp., Coag negStaphylococcus aureus or Nocardia sp. The bacterium may be Legionellapneumophila. The bacterium may be Mycobacterium tuberculosis. The enzymemay be in a cell. The cell may be a mammalian cell.

The present invention provides for a method of selecting a compoundwhich is capable of inhibiting the enzymatic activity of enoyl reductasewhich includes: (A) contacting enoyl reductase with the compound; (B)measuring the enzymatic activity of the enoyl reductase of step (A)compared with the enzymatic activity of enoyl reductase in the absenceof the compound, thereby selecting a compound which is capable ofinhibiting the enzymatic activity of enoyl reductase. The compound maycontact enoyl reductase at same site at which gemfibrozil contacts enoylreductase. U.S. Pat. No. 5,422,372 discloses a method of increasingintracellular accumulation of hydrophilic anionic agents usinggemfibrizol (gemfibrozil). U.S. Pat. No. 4,859,703 discloses lipidregulating compositions. U.S. Pat. No. 4,891,220 discloses a method andcomposition for treating hyperlipidemia. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

Another embodiment of the present invention is a kit which is capable ofdetecting the presence of a particular organism based on the sensitivityof the organism to gemfibrozil. The present invention provides for a kitfor detecting the presence of one or more organisms in a sample whichcomprises: (a) an agar or solution medium suitable for growth of theorganism; (b) a means for testing growth of each organism in thepresence and absence of gemfibrizol such that the growth of the organismor lack thereof can be detected; (c) a means for determining the growthof the organism thus detecting the presence of one or more organisms ina sample. The kit may be in form of an assay, a screening kit or adetection kit.

In one embodiment the compound of the present invention is associatedwith a pharmaceutical carrier which includes a pharmaceuticalcomposition. The pharmaceutical carrier may be a liquid and thepharmaceutical composition would be in the form of a solution. Inanother embodiment, the pharmaceutically acceptable carrier is a solidand the composition is in the form of a powder or tablet. In a furtherembodiment, the pharmaceutical carrier is a gel and the composition isin the form of a suppository or cream. In a further embodiment theactive ingredient may be formulated as a part of a pharmaceuticallyacceptable transdermal patch.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,compression aids, binders or tablet-disintegrating agents; it can alsobe an encapsulating material. In powders, the carrier is a finelydivided solid which is in admixture with the finely divided activeingredient. In tablets, the active ingredient is mixed with a carrierhaving the necessary compression properties in suitable proportions andcompacted in the shape and size desired. The powders and tabletspreferably contain up to 99% of the active ingredient. Suitable solidcarriers include, for example, calcium phosphate, magnesium stearate,talc, sugars, lactose, dextrin, starch, gelatin, cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and Darenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,and oils (e.g. fractionated coconut oil and arachis oil). For parenteraladministration, the carrier can also be an oily ester such as ethyloleate and isopropyl myristate. Sterile liquid carriers are useful insterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellent.

Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by for example, intramuscular, intrathecal,epidural, intraperitoneal or subcutaneous injection. Sterile solutionscan also be administered intravenously. The active ingredient may beprepared as a sterile solid composition which may be dissolved orsuspended at the time of administration using sterile water, saline, orother appropriate sterile injectable medium. Carriers are intended toinclude necessary and inert binders, suspending agents, lubricants,flavorants, sweeteners, preservatives, dyes, and coatings.

The active ingredient can be administered orally in the form of asterile solution or suspension containing other solutes or suspendingagents, for example, enough saline or glucose to make the solutionisotonic, bile salts, acacia, gelatin, sorbitan monoleate, polysorbate80 (oleate esters of sorbitol and its anhydrides copolymerized withethylene oxide) and the like. The active ingredient can also beadministered orally either in liquid or solid composition form.Compositions suitable for oral administration include solid forms, suchas pills, capsules, granulen form. Compositions suitable for oraladministration include solid forms, such as pills, capsules, granules,tablets, and powders, and liquid forms, such as solutions, syrups,elixirs, and suspensions. Forms useful for parenteral administrationinclude sterile solutions, emulsions, and suspensions.

This invention is illustrated in the Experimental Details section whichfollows. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

Experimental Details

The emergence of multiply antibiotic-resistant bacterial pathogens (i.e.M. tuberculosis and S. aureus) has prompted the search for new orunrecognized antibiotic targets in bacteria. Most currently usedantibiotics act by blocking bacterial protein, DNA or RNA synthesis,and/or cell wall assembly. However, as demonstrated by the ability ofisoniazid and ethionamide to inhibit InhA [1,2], an enoyl reductase ofM. tuberculosis [3], bacterial enzymes involved in fatty acid synthesisare also potential antibiotic targets.

While bacterial and mammalian cells use the same general pathways andmechanisms to synthesize fatty acids, bacterial fatty acid synthasesdiffer from their mammalian counterparts in a number of respects. Forexample, mammalian fatty acid synthase is a type I synthase, a homodimercomposed of a single polypeptide encoding seven distinct enzymaticfunctions. Type I synthases perform all of the reactions required forthe synthesis and elongation of fatty acids in mammals [4]. Bacterialfatty acid syntheses are most commonly type II synthases. Type IIsynthases are dissociated fatty acid synthase systems composed ofindividual proteins encoded by distinct genes. Within this system,multiple isozymes of a given protein often exist which catalyze the samebasic chemical reaction but differ in substrate specificity andregulation [5].

Bacteria synthesize many fatty acids not synthesized by human cells(i.e. branched chain fatty acids, di-hydroxy fatty acids). The presenceof these fatty acids is hypothesized to allow bacteria to maintainmembrane fluidity and function upon exposure to a variety ofenvironmental insults including variations in temperature andosmolarity. Drugs that block synthesis of these unique bacterial fattyacids, by inhibiting bacteria-specific enzymes, may block bacterialgrowth without having a detrimental effect on mammalian cells.Accordingly, isoniazid and ethionamide act by inhibiting an enoylreductase involved in the synthesis of mycolic acids, very long chainfatty acids synthesized by M. tuberculosis, but not by human cells.

The findings reported here indicate that gemfibrozil (GFZ), a commonlyprescribed and well-tolerated hypolipidemic agent, inhibits an L.pneumophila enoyl reductase, and has antibiotic activity against a widerspectrum of bacteria than isoniazid. Our findings suggest that bacterialenoyl reductases may be mcre useful targets for novel antibiotics thanpreviously recognized.

Differences in Bacterial and Mammalian Fatty Acid Synthesis

Bacterial fatty acid synthases differ from their mammalian counterpartsin a number of respects, providing potential targets for antimicrobialtherapy. For example, human or mammalian fatty acid synthase (FAS) is aType I synthase. In general, Type I synthases are multifunctionalproteins which perform all or many of the reactions required for thesynthesis and elongation of fatty acids in mammals [4]. In contrast,bacterial FAS are most commonly Type II. Type II syntheses aredissociated systems composed of individual proteins encoded by distinctgenes. Within this system, multiple isozymes of a given protein oftenexist which catalyze the same basic chemical reaction but differ insubstrate specificity and regulation [5].

Not surprisingly, the products synthesized-by bacterial Type IIsyntheses are more varied and complex than those synthesized bymammalian Type I synthases. The end products of Type I mammalian fattyacid synthases are generally palmitate, a sixteen carbon saturated fattyacid (C_(16:0)), myristate (C_(14:0)), and laurate (C_(12:0)). Incontrast, bacterial Type II FAS systems synthesize a complex assortmentof fatty acids the profiles of which can differ greatly among species ofbacteria (FIG. 1). The synthesis of these fatty acids is hypothesized toallow bacteria to maintain constant membrane fluidity and function uponexposure to a variety of environmental pressures including variations intemperature and osmolarity.

The bacterial enzymes involved in the synthesis of these specializedfatty acids generally perform the same basic reactions as thoseperformed by mammalian fatty acid synthases, but have widely differentsubstrate specificities and regulatory characteristics. However, thereare some enzymatic functions which are specific to bacteria includingthe formation of unsaturated fatty acids during elongation, and theformation of cyclopropyl, hydroxylated, and ω-alicyclic fatty acids [8].Drugs that block the synthesis of unique bacterial fatty acids byinhibiting the utilization of bacteria-specific substrates orbacteria-specific enzymes, may block bacterial growth without having adetrimental effect on mammalian cells. Accordingly, the clinicallyeffective and prescribed drugs isoniazid and ethionamide act byinhibiting an enoyl reductase involved in the synthesis of mycolicacids, very long chain fatty acids which are synthesized by M.tuberculosis, but not by human host cells [1].

Fatty Acid Synthesis in Bacteria

Both bacteria and mammalian cells utilize acetyl-CoA as the buildingblock for fatty acid synthesis. Bacteria acquire acetyl-CoA from thedecarboxylation of pyruvate when grown on sugars, and from β-oxidationwhen grown on fatty acids. In mammalian cells, acetyl-CoA is derivedlargely from the tricarboxylic acid (TCA) cycle. High concentrations ofATP and NADH in the mitochondria inhibit the TCA enzyme isocitratedehydrogenase resulting in the accumulation of citrate. Citrate diffusesfrom the mitochondrion into the cytosol where it is converted toacetyl-CoA and then to malonyl-CoA for fatty acid elongation, or toacetyl-ACP for the initiation of fatty acid synthesis.

In E. coli, the initiation or elongation of straight chain fatty acidsynthesis can occur through three different condensation reactions;condensation of an acetyl-CoA with malonyl-ACP, condensation of acyl-ACPwith malonyl-ACP, and the decarboxylation of malonyl-ACP to acetyl-ACPfollowed by condensation with malonyl-ACP. All three condensationreactions form acetoacetyl-ACP (FIG. 2) [5]. The condensation step isthe only irreversible step in fatty acid synthesis in Type II systems.E. coli has three different β-ketoacyl synthases with overlappingsubstrate specificities. However, β-ketoacyl synthase I [9] catalyzes anessential step in unsaturated fatty acid metabolism, that cannot becatalyzed by the other two synthases. β-ketoacyl synthase II [10] isinvolved in the thermal regulation of fatty acid composition, andβ-ketoacyl synthase III [11] catalyzes the initial condensation reactionin the pathway. In mammalian cells, straight chain fatty acid synthesisis initiated through the condensation of acetyl-ACP and malonyl-CoA bythe β-ketoacyl synthase domain of the fatty acid synthase enzyme.

β-ketoacyl synthase activity also mediates the synthesis of branchedchain fatty acids by the condensation of branched chain precursors withmalonyl-CoA at the initiation step of fatty acid synthesis. The primersources for the branched chain fatty acids are generally 2-keto-acidsderived from the branched chain amino acids, valine, leucine, andisoleucine [12,13]. Branched chain fatty acids are synthesized by manyspecies of bacteria and by the sebaceous glands of mammalian skin.

The next step in both straight and branched chain fatty acid elongationinvolves the reduction of the β-ketoacyl-ACP by β-ketoacyl-ACP reductaseto a β-hydroxybutyryl-ACP. Dehydration by β-hydroxyacyl dehydrase yieldscrotonoyl-ACP. This reaction is very inefficient, and the ratio ofsubstrate to product is generally 9:1 [14]. In E. coli, there are twodehydrase enzymes which can catalyze this step [15]. One is involved inthe elongation of saturated fatty acids, and the other serves as thebranch point for the synthesis of unsaturated fatty acids.

The final step in the fatty acid elongation cycle involves the reductionof the enoyl-ACP substrate to generate an acyl-ACP by enoyl reductase.The acyl-ACP is either the end product of fatty acid synthesis, or,serves as the starting material for subsequent cycles of fatty acidelongation. Since the concentration of the enoyl substrate is very low,enoyl reductase is thought to “pull” successive cycles forward. For thisreason it is thought to be the rate-limiting enzyme in fatty acidsynthesis [14].

Inhibition of phospholipid synthesis due to increased levels of thestationary phase alarmone ppGpp in bacteria, leads to the accumulationof acyl-ACPs and inhibition of fatty acid synthesis [16]. In areconstituted fatty acid synthesis assay, the addition of palmitoyl-ACPto the reconstituted system resulted in the accumulation of malonyl ACPand 3-hydroxybutyryl-ACP (3-HB-ACP), presumably by inhibiting of enoylreductase and β-ketoacylsynthase III. However, 3-HB-ACP accumulatedfirst, and at higher concentrations than malonyl-ACP, indicating thatenoyl was the relevant target of palmitoyl-ACP's inhibitory effect [17].

The accumulation of long chain acyl-CoAs occurs during bacterial growthin the presence of long chain fatty acids. Exogenous long chain fattyacids are converted to their CoA thioesters by E. coli, and are eitherused as substrates for β-oxidation, or are preferentially incorporatedinto phospholipids following conversion to their ACP derivatives[18-20]. Long chain acyl-CoAs have been shown to directly inhibit enoylreductase activity and to bind to the global transcriptional regulatorFadR. The interaction of long chain acyl-CoAs (derived from oleate orpalmitate) with FadR releases FadR from DNA, stimulates β-oxidation andfatty acid transport into E. coli, and inhibits three genes involved infatty acid synthesis in these bacteria; fabA, β-hydroxydecenoyldehydrase, the enzyme responsible for unsaturated fatty acid synthesis,fabB, one of three β-ketoacyl synthases, and the enoyl reductase fabI[21-24].

Inhibitors of Fatty Acid Synthesis

Several compounds that inhibit enzymes in fatty acid synthesis have beendescribed. Cerulenin is an inhibitor of fatty acid synthesis inprokaryotes and eukaryotes that acts on β-ketoacyl synthase to inhibitthe condensation of an acyl-ACP or an acetyl-CoA with malonyl-ACP [25].However, since cerulenin inhibits both bacterial and mammalian fattyacid synthesis, it is not clinically useful as an antimicrobial, but isbeing pursued as a chemotherapeutic agent to treat cancers which overexpress FAS [26,27]. Thiolactomycin, is a specific inhibitor of Type IIbacterial β-ketoacyl synthases [28] and is active against many speciesof Gram-positive and Gram-negative bacteria [29]. However, resistance isfrequently acquired [30,31]. 3-decanoyl-N-acetylcysteamine (NAG) is aninhibitor of β-hydroxydecenoyl thioester hydrase, a bacterial enzymecatalyzing the synthesis of unsaturated fatty acids [32,33]. Thiscompound inhibits unsaturated fatty acid synthesis in bacteria includingE. coli, but not in mammalian cells.

Several compounds have been reported to interfere with bacterial enoylreductase activity including isoniazid, ethionamide, triclosan andrelated compounds, and diazoborines Of all the compounds that inhibitenzymes in bacterial fatty acid synthesis, only two, isoniazid andethionamide, are useful as drugs and only for the treatment ofmycobacterial infections [34].

Isoniazid and ethionamide inhibit the InhA enoyl reductase enzyme of M.tuberculosis [1-3,35]. Mutations in InhA, at or near residues involvedin NADH binding, confer resistance to these compounds [1,23] as domutations affecting the intracellular levels of NADH [36]. Isoniazid isa prodrug. Kat G, a catalase-peroxidase enzyme of M. tuberculosis [37]catalyzes the formation of an activated isonicotinic acyl radical whichinteracts with NADH bound at the active site of the InhA enzyme [2]. Thecarbonyl carbon of the isonicotinic acyl group covalently attaches tothe carbon at position four of the nicotinamide ring, replacing the 4Shydrogen of NACH involved in hydride transfer during the reduction of anenoyl substrate. The complex inactivates the enzyme since it displacesthe side chain of Phe¹⁴⁹ allowing it to form an aromatic ring stackinginteraction with the pyridine ring of the isonicotinic group. Thisconformational change increases the affinity of the complex for theenzyme, such that it is not released. Mutations which decrease theaffinity of InhA for NADH may protect the enzyme by promoting thebinding of acyl-ACP substrates before NADH binds. The binding of anacyl-ACP substrate does no allow the bulkier activated isonicotinic acylradical access to the active site.

Isoniazid also has been reported to inhibit the M. tuberculosis fattyacid synthesis β-ketoacyl synthase enzyme, KasA. Mutations in theamino-acid sequence of the KasA protein, were identified inINH-resistant clinical strains of M. tuberculosis that lacked otherknown mutations conferring resistance to INH.

Diazoborines, another group of enoyl reductase inhibitors, exhibitantibacterial activity against most species of Gram-negative bacteria[38] by a similar but distinct mechanism to that of INH. The boron atomin diazoborine forms a covalent bond with the 2-hydroxyl oxygen of thenicotinamide ribose of NADH, generating a bi-substrate analog. Thebicyclic rings of the diazoborines form a face to face interaction withthe nicotinamide ring allowing extensive π-π stacking interactions.Crystallographic studies show that this bi-substrate analog bindsnon-covalently, but tightly, to E. coli FabI enoyl reductase [39],interfering with the access of the reduced pyridine nucleotide (NADH orNADPH) to E. coli FabI's catalytic site. The activity of this class ofcompounds is dependent on the presence of the boron substituent, whichis toxic for mammalian systems [38].

Triclosan, a topical antiseptic, not approved for oral administration,appears to inhibit E. coli enoyl reductases by a mechanism similar tothe diazoborines [40,41]. The phenolic hydroxyl group forms a hydrogenbond (not covalent as for the diazoborines) with the 2-hydroxyl oxygenof the nicotinamide ribose of NADH. The phenol ring of triclosan forms aface to face interaction with the nicotinamide ring allowing extensiveπ-π stacking interactions. Homologous mutations in the E. coli fabI andM. tuberculosis inhA genes, confer resistance to diazoborines,triclosan, or isoniazid, consistent with a NADH—dependent mechanism ofinhibition.

In conclusion, while the basic mechanisms of fatty acid synthesisbetween the mammalian Type I synthases and the bacterial Type IIsynthases are conserved, significant differences exist. Thesedifferences should be exploitable for the creation of new classes ofantibiotics. The need for antibiotics that inhibit bacterial growth bymechanisms other than those used by current antibiotics is increasing asthe number of bacterial species resistant to multiple drugs grows. Thefindings that isoniazid and ethionamide [1], the diazoborines [42], andtriclosan [40] all act by inhibiting enoyl reductases suggest that thiskey regulatory enzyme in fatty acid biosynthesis is an excellentantimicrobial target.

GFZ Inhibits the Growrh of Legionella Pneumophila in Macrophages and inNutrient Broth

Gemfibrozil (Lopid™) is well known as a hypolipidemic agent that lowersLDL and triglyceride levels in humans. The mechanism(s) by which GFZexerts this effect is unresolved. GFZ has also been reported to inhibitorganic anion transport in mouse J774 macrophages [431]. Although theendogenous substrates for this transporter have not been identified, itis known that anionic compounds, including Lucifer Yellow, fluorescein,penicillin and the fluoroquinolone antibiotics ciprofloxacin andnorfloxacin, are efficiently secreted by J774 macrophages by GFZinhibitable transporters [43-46].

Inhibitors of anion efflux should increase the intracellularconcentration of anionic antibiotics, thus increasing the efficacy of agiven oral or intravenous dose for intracellular pathogens. Addition ofGFZ in combination with norfloxacin, reduced by fourfold theconcentration of norfloxacin required to block intracellular growth ofListeria monocytogenes in mouse J774 macrophage-like cells [46]. Thiswas consistent with previous findings in which treatment of J774 cellswith GFZ increased the intracellular concentration of norfloxacin in theJ774 cells fourfold [44].

L. mohocytogenes grows in the cytoplasm of macrophages, Otherintracellular pathogens reside in specialized membrane-boundintracellular compartments. For such pathogens, increasing theconcentration of antibiotics in the cytosol may have no effect if theconcentration of the antibiotic is not increased in thepathogen-containing compartment. Alternatively, if the antibioticreadily penetrates the pathogen-containing compartment, then increasesin the cytoplasmic concentration of the given antibiotics shouldpotentiate the antimicrobial effect of the antibiotic. Since GFZ exertsthe latter effect on fluoroquinolone antibiotics it was desirable toevaluate the effect of GFZ in combination with these antibiotics againstintracellular pathogens that grew within membrane-bound compartments inmacrophages. We began with Legionella pneumophila, an intracellularpathogen responsible for up to 15 percent of all community-acquiredpneumonias requiring hospitalization [47].

L. pneumophila is an environmental pathogen most commonly found in watersources such as shower heads, water towers and air conditioningcondensers. Aerosolization of contaminated water sources allows thebacteria to be inhaled into the lungs where it infects alveolarmacrophages. L. pneumophila enters macrophages within phagosomesproduced as a result of a process known as coiling phagocytosis }[48].The Legionella-containing phagosomes go through a unique series ofmodifications such that acidification is avoided, and mitochondria,smooth vesicles, ribosomes and rough endoplasmic reticulum are recruitedto their periphery [49-52]. In human macrophages, or in themacrophage-like cells of the HL-60 human myelocytic cell line, bacterialreplication generally begins within eight hours of bacterial uptake[52,53]. Twenty four to thirty six hours after infection, the cellsround up, undergo either bacterially induced lysis or apoptosis [53-55],and release the expanded population of intracellular bacteria forsubsequent rounds of infection.

While testing the ability of GFZ to increase the efficacy offluoroquinolone antibiotics in a L. pneuniophila infection model, Idiscovered that GFZ alone inhibited the intracellular growth of L.pneumophila in peripheral-blood monocyte derived human macrophages andin the phorbol myristate acetate (PMA)-differentiated macrophage-likecells of the HL-60 human promyelocytic cell line.

Results: Addition of GFZ to a final concentration of 0.4 mM (100 μg/ml)to the medium of PMA-differentiated HL-60 cells infected with L.pneumophila resulted in inhibition of L. pneumophila intracellulargrowth (FIG. 3). To determine whether growth inhibition was due to acytotoxic effect of GFZ on the HL-60 cells, cellular viability assayswere performed on uninfected and infected HL-60 cells. The MTT assay wasused, which assesses cellular respiration via the reduction of atetrazolium dye to an insoluble blue formazan in viable cells [53,56,57]to test HL-60 viability. GFZ at 100 μg/ml did not affect the viabilityof uninfected HL-60 cells, even when the cells were incubated or fivedays. Furthermore, the presence of GFZ in the medium protected infectedHL-60 cells from L. pneumophila's cytolytic effects even at an infectionmultiplicity of 0.1 bacteria/cell (FIG. 4). Therefore, it was possiblethat GFZ was directly inhibiting the growth of L. pneumophila.

To assess whether GFZ had the potential to directly inhibit L.pneumophila growth, a minimum inhibitory concentration (MIC) assay wasperformed. L. pneumophila was added to. ACES-buffered yeast extract(AYE) liquid medium containing varying concentrations of GFZ. The MICwhich inhibited 90% of L. pneumophila growth was 10 μg/ml of GFZ. At allconcentrations tested (10-200 μg/ml), GFZ was bacteriostatic for L.pneumophila Philadelphia 1, as determined by plating 5 μl aliquots ofthe MIC cultures on charcoal yeast extract (CYE) and visually assessinggrowth after four days of incubation at 37° C.

L. pneumophila does not grow in human plasma or in mammalian tissueculture medium so it was unlikely that GFZ blocked L. pneumophila growthby affecting extracellular bacteria. Nonetheless, a single-round growthassay was developed to confirm that GFZ had access to, and wasinhibiting the growth of intracellular L. pneumophila within HL-60cells. In this assay gentamicin was added to the medium two hours afterL. pneumophila infection to kill any remaining extracellular bacteria.After a 30 minute incubation, the gentamicin-containing medium waswashed away and replaced with fresh medium containing GFZ at a finalconcentration of 0.4 mM (100 μg/ml). Inhibition of bacterial growth wasseen within 8 hours after infection, well within the 24-36 hour timeperiod required for L. pneumophila to lyse these cells [50,54,55] (FIG.5).

This experiment shows that GFZ inhibits the growth of L. pneumophilaintracellularly. L. pneumophila also grows intracellularly in primaryhuman blood monocyte-derived macrophages [54,58]. In accordance with theresults found in the HL-60 cell line, GFZ at a concentration of 0.4 mM(100 μg/ml), inhibited the intracellular growth of L. pneumophila inhuman macrophages (FIG. 6). GFZ at 0.4 mM was optimal since no furtherreduction in L. pneumophila colonies was observed with GFZ 0.6 mM.

Although the previous experiments demonstrated that growth inhibitionwas not due to a cytotoxic effect in HL-60 cells, it was still possiblethat GFZ was inhibiting growth through a “fibric acid-mediated” effecton the macrophages. GFZ belongs to a class of fibric acids includingclofibrate, fenofibrate, and bezafibrate, all of which lower serumtriglyceride levels though an unresolved mechanism. These analogs arealso known to stimulate peroxisome proliferator activated receptors(PPARs) in rats, although this effect does not occur in humans. PPARsare transcriptional activators which affect genes in several metabolicpathways including cholesterol biosynthesis [59-61] and the β-oxidationof fatty acids [62,63].

To test whether GFZ inhibition of intracellular L. pneumophila growthoccurred through a fibric acid mediated effect on host cell geneexpression, other fibric acids were examined (FIG. 6). When bezafibratewas added to human macrophages infected with L. pneumophila at aconcentration of 1 mM, no inhibition of L. pneumophila growth wasobserved. Clofibrate, another structural analog of GFZ (FIG. 7), wasable to slow L. pneumophila growth although a concentration of 1 mM wasrequired. Fenofibrate precipitated out of solution at a concentration of0.1 mM, so it was not studied in this system. The ability of thesefibric acids to inhibit L. pneumophila growth in AYE broth was assessedby MIC assays. While GFZ has an MIC of 10 μg/ml, clofibrate was found tohave an MIC of 100 μg/ml, and bezafibrate an MIC of 250 μg/ml.Fenofibrate precipitated out of solution at a concentrations above 100μg/ml, and did not inhibit L. pneumophila growth at this concentration.

This is the first report of GFZ's antibiotic activity. It was highlysurprising and serendipitous to find that a compound used safely in manfor over twenty years to lower triglycerides, inhibited bacterialgrowth. This suggested that GFZ might be inhibiting a bacterial targetwhich is not expressed in humans or is resistant to GFZ in humans.

The mechanism by which GFZ exerts its hypolipidemic effect is unknown.At least seven different pathways in lipid metabolism have been shown tobe affected by GFZ, either in vivo or in cell culture. The sevenpathways affected by GFZ are: 1) GFZ stimulates lipoprotein lipaseactivity, cleaving fatty acids from triglycerides in the VLDL fractionof the plasma; 2) GFZ increases the activity of lipoprotein lipase[61,64]; 3) GFZ stimulates intracellular triglyceride synthesis [65]possibly mediated through an increase in acyl-CoA synthetase expression[66]; 4) GFZ promotes the transcription of several enzymes involved inthe β-oxidation of fatty acids through the stimulation of peroxisomeproliferator activated receptors (PPARs), although peroxisomeproliferation has only been demonstrated in rats [62,63]; 5) GFZdecreases microsomal fatty acid elongation [67,68]; 6) GFZ stimulatesthe synthesis of Apo A-1, the major protein in HDL [59,69] and 7) GFZdecreases the synthesis of cholesterol [60]. Any or all of thesemechanisms could be involved in lowering serum lipids.

Although GFZ inhibits intracellular L. pneumophila growth withinmacrophages, the concentration required to inhibit growth within thesecells (100 μg/ml) is ten-fold higher than that required to inhibitgrowth in AYE broth (10 μg/ml). The finding that a lower concentrationof an antibiotic is required to inhibit extracellular versusintracellular growth of a bacterial pathogen is not uncommon. Otherantibiotics which inhibit L. pneumophila growth, both extracellularlyand intracellularly are erythromycin, doxycycline, and rifampin. Allthree of these antibiotics inhibit extracellular growth of L.pneumophila at lower concentrations than intracellular growth (e.g.erythromycin at 0.2 μg/ml versus 1.25 μg/ml, doxycycline at 0.4 μg/mlversus 0.8 μg/ml, and rifampin at 0.002 μg/ml versus 0.1 μg/ml [70-73].For an antibiotic to inhibit the intracellular growth of a bacterialpathogen, it must enter the host cell, enter the compartment thatcontains the pathogen, and be retained within that compartment in anactive form [74].

Therefore, with respect to L. pneumophila, several explanations couldaccount for the difference in MICs. First, in work not described here,the addition of 10% serum to AYE increased GFZ's MIC from 10 μg/ml to 30μg/ml, suggesting that serum contains factors capable of binding GFZ,thereby reducing its effective concentration. Charcoal is known to havea similar effect such that the MICs of antibiotics for L. pneumophila onACES-buffered charcoal yeast extract (ABCYE) agar [75], range from twoto twenty fold higher than the MIC values found on buffered potatostarch yeast extract (BSYE) agar. The latter contains potato starchinstead of charcoal [76]. Second, GFZ may be transported out of the hostcell cytoplasm, or, out of the ribosome-studded compartment in which L.pneumophila grows, thus limiting the amount available to inhibitintracellular L. pneumophila. Third, the host cell may metabolize GFZ toeither a less active compound, or, a compound that has limited access tothe L. pneumophila-containing compartment. Fourth, L. pneumophila may bemore resistant to GFZ when growing intracellularly. Barker et al.reported that intracellular growth enhances resistance to antibiotics inL. pneumophila [77]. Fifth, metabolites provided by the host cell mayreduce GFZ's inhibitory effect.

We draw several conclusions from these experiments. First, a fibric acidmediated effect on host cell transcription is not likely to be the causeof GFZ's ability to inhibit the intracellular growth of L. pneumophilasince bezafibrate was totally ineffective and clofibrate much lesseffective than GFZ at inhibiting intracellular growth (FIG. 6). Second,the ability of GFZ to inhibit the growth of L. pneumophila in AYE brothat a concentration of 10 μg/ml demonstrates that GFZ has a directinhibitory effect on L. pneumophila. The ability of GFZ to inhibitintracellular growth strongly suggests that GEL has access to the uniqueintracellular compartment in which L. pneumophila resides. Further workwith ³H-GFZ is required to confirm this suggestion. Third, metabolitesprovided by the macrophages do not circumvent the capacity of GFZ toinhibit the intracellular growth of L. pneumophila.

While many potential antibiotics are often highly effective againstpathogens in laboratory conditions, they often fail in animal infectionmodels and/or clinical trials due to unexpected toxicity, or a lack ofeffect, in a mammalian host. Since GFZ has been used therapeutically foryears, there is a large body of literature confirming its safety, evenat much higher levels that those used to treat hyperlipidemia [78,79].

In humans, GFZ is reported to achieve a serum concentration of 12-15μg/ml within 2 hours following a single oral dose of 450-600 mg [78,79].These levels were sustained for an additional four hours, but fell to 5μg/ml by 9 hours after administration [78-81]. Although the MIC (10μg/ml) for L. pneumophila in nutrient broth is within the serum levelsachieved for the treatment of hypertriglyceridemia, a concentration of100 μg/ml was required to inhibit intracellular L. pneumophila growth.However, plasma drug levels are proportional to the dose [82], so aserum GFZ concentration of 100 μg/ml could be achieved through higherdosage. A serum concentration of 100 μg/ml in mice would only require3.24 mg/mouse per day (1.08 mg every eight hours) well below the LD₅₀ of250 mg/20 gm mouse per day for mice [78,79].

In summary, these experiments revealed that GFZ is inhibitory for L.pneumophila growth in AYE broth and within macrophages. This finding hasrelevance for the use of GFZ to treat bacterial infection in humanssince GFZ is known to have relatively low toxicity in humans, even withlong term use as a therapeutic agent for hypertriglyceridemia. Furtherstudies are needed to assess the potential therapeutic use of GFZ, orrelated analogs, in the treatment of human bacterial infections.

Methods and Materials

Bacterial and Growth Conditions. L. pneumophilia Philadelphia 1serogroup 1 was a generous gift from Dr. Marcus Horowitz [53]. L.pneumophila was grown in ACES-buffered yeast extract (AYE) broth [71]that lacked bovine serum albumin at 37° C. with aeration, or, onN-[2-acetomido]-2-aminoethane sulfonic acid (ACES)-buffered charcoalyeast extract (ABCYE) agar plates [75], at 37° C. in the presence orabsence of GFZ. GFZ and ACES were purchased from Sigma. All other mediacomponents were purchased from Fisher.

HL-60 Intracellular L. pneumophila inhibition assays. Tissue culturemedium Roswell Park memorial Institute (RPMI) 1640 tisue culture mediumwas obtained from JRH Biosciences, Lenexa, KS; L-glutamine (GLN) fromMediatech, Herndon, Va., and phorbol 12-myristate 13-acetate (PMA) fromSigma. and normal human serum (NHS) (Ultraserum™) from GeminiBio-Products, Calabasas, Calif. Promyelocytic HL-60 cells weredifferentiated into macrophage-like cells by incubation with 10 ng/mlPMA in RPMI with 2 mM GLN and 10% NHS in Teflon wells at 37° C. for 24hours. These cells were washed, resuspended at 4×10⁶ cells/ml in RPMI-2mM GLN-10% NHS, and mixed with L. pneumophila Phil 1 (4×10⁴ CFU/ml)which had been grown for 2 days on ABCYE plates (final multiplicity ofinfection of 0.01). 100 μl aliquots of the suspension were plated ineach well of a 96 well microtiter plate. The plates were centrifuged topellet the cells and bacteria and incubated at 37° C. for 2.5 hours toallow phagocytosis of the L. pneumophila. 100 μl of fresh medium with orwithout 2× the final GFZ concentration, was added to the wells, and theplates were incubated at 37° C. At the times indicated, the cells andmedium were harvested and assayed for L. pneumophila CFUs as described[58].

For the one-step assay, following pelleting of HL-60 cells and bacteria,the plates were incubated for 2.5 hrs to allow the HL-60 cells tointernalize the bacteria. Gentamicin (100 μg/ml) was added for 0.5 hrsto kill extracellular bacteria. The cells were washed to removegentamicin, overlaid with 200 μl fresh RPMI-2 mM GLN-10% NHS without orwith GFZ (100 μg/ml), and incubated at 37° C. At the times indicated,the cells and medium were harvested and assayed for L. pneumophila CFUsas described [58]. L. pneumophila is unable to grow in tissue culturemedium, so any increase in colony forming units reflects intracellularmultiplication [54]. Data from the experiments are expressed as theaverage (+/−) the S.E.M (n=3).

HL-60 cytotoxicity assay. HL-60 cells (4×10⁵ cells per well) weredifferentiated in the wells of a 96 well microtiter dish by incubationfor two days at 37° C. in an atmosphere of 95% air 5% CO₂ with 10 ng/mlPMA in RPMI-2 mM GLN-10% NHS. Adherent cells were washed two times withRPMI-2 mM GLN and then incubated with RPMI-2 mM GLN-10% NHS (+/−) 100μg/ml GFZ. 5-fold serial dilutions of L. pneumophila in RPMI, were addedto the wells at multiplicities of 0.5, 0.1, 0.02, 0.004, and 0.0,starting with 2×10⁵ bacteria added per well. After a 5 day incubation at37° C., the dye MTT ((3-4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma), was added to each well at a concentrationof 500 μg/ml. In this assay the A₅₇₀ is proportional to the number ofviable macrophages in the wells [58]. The microtiter dishes wereincubated for 4 hrs at 37° C., the culture medium was aspirated, and thereduced formazan dye was suspended in 100 μl of 0.04 M HCl-1% sodiumdodecyl sulfate in isopropanol. The A₅₇₀ values of six separate wells,that had been seeded with 4×10⁵ HL-60 cells infected with L. pneumophilainfected at the same multiplicity of infection and incubated with orwithout GFZ, were averaged to determine macrophage viability.

Human Peripheral Blood Monocyte-derived Macrophage Assays

Human leukocytes were purified by layering buffy coats onHistopaque-1077 and centrifuging at 400×g for 15 min. The leukocytelayer was washed 3× in RPMI 1640-2 mM GLN, resuspended in RPMI 1640-2 mMGLN-20% heat-inactivated human serum, added to 75 cm² flasks andincubated at 37° C. in an atmosphere of 95% air, 5% CO₂ to allowadhesion of monocytes. After two hours the nonadherent cells wereremoved by washing and fresh RPMI 1640-2 mM GLN-20% heat-inactivatedhuman serum, was added to the adherent monolayers. Following 12 hoursincubation, the medium was replaced with PD buffer containing 5 mM EDTA,and the flasks were incubated at 37° C. 5% CO₂ for 30 minutes to detachthe monocytes. The detached monocytes were pelleted at 400×g for 15minutes, and resuspended in RPMI 1640-2 mM GLN +30% heat-inactivatedhuman serum, and stored in Teflon wells at 37° C. in humidifiedincubators in an atmosphere of 95% air and 5% CO₂. Human monocytederived macrophages maintained in culture for five days were resuspendedin fresh RPMI 1640-2 mM GLN+10% normal human ultraserum containing L.pneumophila at an MOI of approximately 0.001. 100 μl aliquots containing4×10⁵ monocyte-derived macrophages and 1×10² L. pneumophila werepelleted in the wells of a 96 well microtiter plate and incubated at 37°C. for 2.5 hours to allow phagocytosis of the L. pneumophila. 100 μl offresh medium with or without 2× the final concentration of GFZ,clofibrate or fenofibrate, was added to each well. The plates wereincubated at 37° C. in humidified incubators in an atmosphere of 95% airand 5% CO₂. At the indicated times, the cells and medium were harvestedand assayed for L. neumophila CFU's.

Antimicrobial susceptibility testing. For determination of MICs,triplicate cultures of log phase L. pneumophila suspensions, finalconcentration 2×10⁶ CFU/ml, were incubated in AYE broth containingtwo-fold serial dilutions of GFZ, fenofibrate, clofibrate, orbezafibrate for 48 hrs at 37° C. Growth was assessed by the opticaldensity at A₆₀₀. Bacteriostatic effect was determined by incubating L.pneumophila suspensions, final concentration 2×10⁶ CFU/ml, in AYE brothcontaining two-fold serial dilutions of GFZ (10-200 μg/ml) for 48 hrs at37° C. 5 μl from each culture was spotted on ABCYE agar and incubated at37° C. for three days. L. pneumophila grew in all spots indicating thatGFZ was bacteriostatic, not bacteriocidal.

GFZ Inhibits the Growth of Mycobacterium Tuberculosis and OtherPathogens

Gemfibrozil, Lopid™, a compound prescribed for hypertriglyceridemia inhumans, was discovered to be an inhibitor of L. pneumophila growth inAYE broth (MIC₉₀=10 μg/ml) and in macrophages (100 μg/ml). The discoverythat gemfibrozil (GFZ) inhibited the growth of L. pneumophila suggestedthat GFZ might inhibit additional bacterial species. GFZ demonstratedactivity against 33% of the bacteria screened, including Mycobacteriumtuberculosis, Nocardia sp., Staphylococcus aureus, and Staphylococcusepidermidis. Two yeast species, Sacchromyces cerevisiae and Candidaalbicans were also found to be susceptible to GFZ.

The susceptibility of M. tuberculosis was of particular interest sinceM. tuberculosis claims more lives, roughly 3 million people per year,than any other single infectious disease in the world [84]. While thereported number of new cases of tuberculosis in the United States isdeclining [85], it is nearly impossible to eradicate the disease sinceit can remain dormant and undetected in immunocompetent hosts for years.On average at least 5% of immunocompetent hosts will develop activedisease in their lifetimes [86]. The rate is significantly higher forthose who are, or become, immunocompromised [87,88].

Despite the magnitude of the problem, no new primary anti-tuberculosismedicines have been developed since the 1960's [89]. The emergence andrapid spread of multiple drug resistant M. tuberculosis strains has ledto renewed interest in the development of compounds to treat and controlthis deadly organism. Unfortunately, significant mortality due tomultiple drug resistant bacteria may no longer be limited to M.tuberculosis.

RESULTS

Screening of Various Species of Bacteria and Yeast for GFZ-mediatedGrowth Inhibition

L. pneumophila Philadelphia 1 serogroup 1 was sensitive to growthinhibition by GFZ. To determine whether GFZ was specific for this strainof L. pneumophila we tested its effect on 38 other Legionella sp.strains using a zone of inhibition assay. A disk containing 250 μg ofGFZ was added to a CYE agar plate overlaid with the test bacterium. Theabsence of bacterial growth in the area adjacent to the disk, or a “zoneof inhibition,” indicated sensitivity to GFZ. All 39 Legionella sp.strains were sensitive by this assay.

Additional nonpathogenic bacterial species were obtained from Dr. DavidFigurski and tested with the same assay. The finding that four of theeight strains tested demonstrated sensitivity to GFZ led to acollaboration with the Clinical Microbiology Department of PresbyterianHospital to screen randomly-selected clinical strains of bacterial andfungal pathogens. The screen was performed by adding a sterile diskcontaining 2 mg of GFZ to a nutrient agar plate overlaid with the testpathogen. Eleven of the thirty one bacterial species tested, or 33%,demonstrated susceptibility to GFZ (FIG. 8). A variation of this assaywas utilized for screening the mycobacterial strains in that diskscontaining 2 mg GFZ were embedded in 5 mls of nutrient agar prior tooverlaying with bacteria.

Human pathogens in the susceptible group in addition to L. pneumophilaincluded M. tuberculosis, Nocardia sp., S. epidermidis, and S. aureus.All of the susceptible species are reported to contain branched chainfatty acids }[13], although not all bacteria containing branched chainfatty acids demonstrated susceptibility to GFZ (e.g. L. monocytogenes)[13,46].

S. aureus and S. epidermidis susceptibility to GFZ on standardlaboratory medium was fairly low, only a narrow zone of inhibition wasobserved (e.g. 2-5 mm). To see if nutrients supplied by the medium might“rescue” S. aureus and S. epidermidis from the effects of GFZ, fourstrains of S. aureus and one strain of S. epidermidis were each platedon LB, a nutrient-rich medium, and TSB, a relatively nutrient-pouomedium. The zones of inhibition were significantly larger on the TSBplates (e.g. 10-20 mm) (FIG. 9).

Nocardia sp. susceptibility was notable in that GFZ produced large zonesof inhibition, e.g. 40-60 mm, by the disk assay. It was also noted thatthe GFZ zone of inhibition assay appeared to be an effective method ofrapidly differentiating Nocardia sp. from atypical mycobacteria, all ofwhich were resistant to GFZ on standard laboratory media.

Saccharomyces cerevisiae and Candida albicans were also found to besusceptible to GFZ when grown on SAB medium buffered to a pH of 7. Nozone of inhibition was observed with unbuffered medium, as GFZ isinsoluble at an acid pH.

MIC Determination for M. Tuberculosis

The susceptibility of M. tuberculosis to GFZ was of special interestgiven the prevalence, morbidity, and mortality associated withinfections by this organism. Therefore, we tested the GFZ susceptibilityof 27 M. tuberculosis strains, 22 of which were resistant to one or moreanti-tubercular drugs, by plating M. tuberculosis strains on nutrientmedium containing 0, 50, 100, or 200 μg/ml of GFZ. Growth of all M.tuberculosis strains was completely inhibited by 100-200 μg/ml GFZ,regardless of their resistance to other antibiotics (FIG. 10).Comparable results were obtained when M. tuberculosis was added to 7H9broth containing GFZ at concentrations of 50 or 300 μg/ml (FIG. 11).

EMS Mutagenesis

To search for genes involved in GFZ susceptibility/resistance, andthereby identify its mechanism of inhibition, GFZ resistant mutants ofL. pneumophila were sought. Over 10¹² CFUs of wild type orEMS-mutagenized L. pneumophila were plated on CYE plates containing 50μg/ml of GFZ. No spontaneous mutants were obtained. Only oneEMS-mutagenized L. pneumophila variant, F4b, was identified. F4b had anMIC₉₀ of 50 μg/ml in an AYE MIC assay, compared to 10 μg/ml GFZ for thewild type L. pneumophila parent strain. F4b also demonstrated increasedresistance to GFZ in an intracellular infection assay in HL-60 cells(FIG. 12).

GFZ Analog Assays

Previous results from the intracellular and broth assays testing theeffect of fibric acids on L. pneumophila, indicated that structuralanalogs of GFZ, such as clofibric acid, had a modest inhibitory effecton L. pneumophila growth. Therefore, several GFZ analogs were obtained(FIG. 13) and tested by a zone of inhibition assay against L.pneumophila and the partially GFZ-resistant variant F4b (FIG. 14). Twoof the analogs, 4-HPA and 3,4-HPA, were equally inhibitory for both wildtype L. pneumophila and the partially GFZ- resistant variant F4b. Ofnote was the finding that both wild type L pneumophila and the F4bvariant were more sensitive to 2-hydroxybenzoic acid (salicylic acid)than GFZ. Aside from GFZ, F4b demonstrated increased resistance to2-hydroxybenzoic acid, also known as salicylate.

The random bacterial screen demonstrated that GFZ was active against anumber of bacteria, notably M. tuberculosis, Nocardia sp. S. aureus andS. epidermidis all of which can cause infections which may be difficultto treat. M. tuberculosis, an acid-fast bacillus, latently infects up toone third of the world's population and is the leading cause of death inhumans from a single infectious agent [84]. M. tuberculosis primarilyinfects the lungs, although extrapulmonary infection, with disseminationthroughout the body also occurs.

Nocardia sp. are gram-positive, weakly acid-fast, higher bacteria, also-known as actinomycetes, that form branches similar to fungii, butinduce a neutrophilic inflammatory response similar to other bacteria,and demonstrate susceptibility to antibiotics. Nocardia sp. primarilyinfects the lungs, resulting in a pneumonia but can spread to the skinand brain where it forms abscesses. Six to twelve months of antibiotictreatment may be required to cure Nocardia infections.

S. aureus, a gram-positive clustering coccus, often colonizes the nose,and is responsible for numerous serious infections includingendocarditis, osteomyelitis, toxic shock syndrome, and pneumonia.Strains of this organism have been identified that are resistant to allknown antibiotics. S. epidermidis is the primary agent associated withinfections of prosthetic devices including heart valves, hip and jointreplacements, and indwelling catheter lines. S. epidermidis is alsoassociated with urinary tract infections in sexually active women.

L. pneumophila, a gram-negative bacilli, is the third or fourth mostcommon cause of community-acquired pneumonia, and is a frequent cause ofnosocomial pneumonia. Infection with L. pneumophila is associated withsignificant morbidity, mortality, and hospital costs.

C. albicans, a pathogenic fungi, is associated with oral, esophageal,intestinal, and vaginal infections in immunosuppressed patients, andcommonly with vaginal infections in an immunocompetent population.

The observation that all susceptible bacteria (and yeast) containbranched fatty acids in their membranes indicates that GFZ may beinhibitory for bacteria capable of synthesizing a complex array ofmembrane fatty acids. dditional support for the involvement of fattyacids in GFZ inhibition is provided by the observation that addition ofoleate to the medium of GFZ-inhibited S. cerevisiae, rescues these yeastfrom the inhibitory effects of GFZ. Addition of oleate to the mediumalso rescues S. cerevisiae from the antibiotic cerulenin, an inhibitorof fatty acid synthesis in yeast, bacteria, and mammalian cells [90.91].

Similarly, growth inhibition in E. coli mediated by drugs or by tsmutations affecting fatty acid synthesis, can be bypassed in many casesby the addition of fatty acids to the bacteriologic medium such as oleicacid (C_(18:1)), a mono-unsaturated sixteen carbon fatty acid, andpalmitic acid (C_(16:0)), a saturated sixteen carbon fatty acid [32,92].The observation that S. aureus showed increased susceptibility to GFZ onTSB medium as compared to the nutrient rich LB medium, suggests that LBsupplies metabolite(s), possibly fatty acids, that are able to bypassthe effect of GFZ.

It is important to note that the M. tuberculosis assays were performedin the presence of oleic acid. Standard mycobacterial medium utilizesoleic acid (or Triton) as a detergent to prevent “cording” of thebacteria. It is possible that if oleic acid is left out of the medium,M. tuberculosis will exhibit greater susceptibility to GFZ. However,testing the ability of GFZ to inhibit the growth of M. tuberculosiswithin infected human macrophages may be a more relevant approach.

Therefore, if the bacterial strains screened for GFZ-susceptibility(FIG. 9) were re-tested on medium lacking fatty acids, additionalsusceptible strains might be identified. The GFZ-sensitivity of bacteriatested on nutrient agar free of fatty acids may better correlate withthe susceptibility of these bacteria to GFZ in vivo.

Other factors besides the presence of fatty acids may contribute to thepresence or size of a zone of inhibition adjacent to a GFZ disk. Theability to observe a zone of inhibition for S. cerevisiae and C.albicans was dependent on buffering the medium to a pH of 7. GFZsolubility in aqueous solution decreases as pH decreases. Therefore, toensure diffusion of the drug through the medium, and to preventacidification of the medium during growth, it was necessary to bufferthe pH. The effects of pH on GFZ sensitivity was not examined with anyother medium or pathogen.

The zone of inhibition surrounding a GFZ disk represents the area inwhich the concentration of GFZ is high enough to inhibit bacterialgrowth. If GFZ is not very soluble in a given medium, or is tightlybound by proteins in the medium (e.g. albumin), the rate of diffusionfrom the disk may be slowed, resulting in a short and steepconcentration gradient. The rate of diffusion is also affected by thethickness of the agar plate since the drug diffuses in three dimensionalin agar, i.e., the thicker the plate, the smaller the zone. Nonetheless,for screening purposes, zones of inhibition afford a rapid and easymethod by which to assess the presence, but not the extent, of GFZsensitivity.

The observation that M. tuberculosis strains that are resistant tomultiple conventionally used antibiotics were as sensitive to GFZ as M.tuberculosis. strains that are sensitive to these antibiotics suggeststhat GFZ may be a lead compound for identifying antibiotics that caninhibit the growth of multiply drug resistant M. tuberculosis. Therelative impermeability of the cell well accounts for the majority ofthe drug resistance in M. tuberculosis strains. However, many potentialdrug resistance determinants, including β-lactamases, aminoglycosideacetyl transferases, and many potential drug efflux systems, are encodedin its genome [93]. Whether any of the chromosomally encoded potentialdrug resistance determinants confers increased resistance to GFZ isunknown.

Resistance to drugs can occur by several mechanisms. Drug resistance mayresult from the overuse of a drug, thereby selecting for organisms thatgrow despite the presence of the drug. The sporadic use of drugs, whichoften happens in unobserved TB therapy, selects for increasinglyresistant bacterial populations. Subinhibitory concentrations of a drugalso enhance the outgrowth of drug-resistant mutant strains, andencourage the spread of plasmids encoding drug resistance mechanismsfrom one species to another. Unfortunately, the development ofresistance to one drug, often confers resistance to other drugs withinthe same class.

Our inability to identify spontaneous or EMS-generated GFZ resistantmutants suggests either that the L. pneumophila target cannot be readilyaltered to confer resistance to this drug, or, that GFZ can affect morethan one enzyme or pathway in L. pneumophila. The ability to inhibitmultiple targets in an organism is a desirable property for anantibiotic.

The observation that GFZ inhibited only 33% of the bacteria screenedsuggests that GFZ has a narrow spectrum of antimicrobial activity. Whilenarrow spectrum antibiotics are less likely to generate the revenuesdesired by large pharmaceutical companies, they have the potentialadvantage of targeting the primary agent of disease without inhibitingthe body's normal flora. Antibiotics with these characteristics shouldlimiting the generation and spread of drug resistant bacteria. Thefinding that GFZ inhibited twenty three drug resistant strains of M.tuberculosis, Nocardia sp. and S. aureus, encouraged us to continueresearch in this area, especially with regard to the mechanism(s) ofGFZ-mediated bacterial inhibition.

Materials and Methods

Antimicrobial Susceptibility Testing

For determination of bacterial susceptibility, bacterial suspensionswere overlaid on suitable nutrient agar plates, a sterile paper diskcontaining 2 mg of GFZ was added, and the plates were incubated at 37°C. until a lawn of bacterial growth was observed. Bacteria wereclassified as susceptible if there was a zone of growth inhibition ofmore than 2 mm surrounding the disk. Non-pathogenic bacterial strainswere graciously provided by D. Figurski, College of Physicians andSurgeons, Columbia University, NYC, N.Y. Pathogenic bacterial strains(except for L. pneumophila) used for GFZ susceptibility testing wereclinical isolates obtained from Dr. P. Della Latta, Director ClinicalMicrobiology, Presbyterian Hospital, and generously screened followingNCCLS standardized procedures by the Clinical Microbiology Dept. ofColumbia-Presbyterian Hospital . A sterile disk containing 2 mg ofgemfibrozil (Sigma) was added to the overlay. Sensitivity was determinedby the presence of a zone of growth inhibition surrounding the disk.

M. tuberculosis susceptibility assay. 100 μl of a standard dilution ofeach of 5 drug-sensitive and 22 drug-resistant M. tuberculosis strainswere tested for GFZ-susceptibility on 5 ml quadrants of solidMiddlebrook 7H10 medium (Baltimore Biological Labs) supplemented witholeic acid, dextrose, catalase, and albumin (OADC) obtained from Difco,Detroit, Mich., containing 0, 50, 100, or 200 μg/ml of GFZ. Standarddilutions were prepared by resuspending each strain to a McFarlandstandard of one, approximately 10⁸ organisms, and diluted 100-fold.Strains were obtained from the Clinical Microbiology Dept. ofColumbia-Presbyterian Hospital. Plates were scored following incubationfor three weeks at 37° C. For broth assays, approximately 10⁷ bacteria(100 μl of a Mc Farland standard of 1) was added to BBL Prepared CultureMedia (Beckton Dickenson) containing 5 mls of Middlebrook 7H9 broth withglycerol. The cultures were incubated for 21 days at 37° C. after whichturbidity was visually assessed for growth.

EMS mutagenesis. Log phase L. pneumophila in AYE broth were incubatedwith 15 μl EMS for 15 minutes at 37° C., pelleted, washed twice,resuspended in 1 ml AYE, titered for CFUs, diluted 1:10 in AYE and grownovernight at 37° C. EMS exposure resulted in approximately 40% and 70%viability in two independent experiments. Following overnight growth,the EMS-mutagenized L. pneumophila were grown on CYE plates at 37° C.for two days, harvested, and suspended at approximately 10¹⁰ CFU's/ml inAYE broth. 100 μl aliquots were plated on CYE agar containing 50 μg/mlGFZ. Putative GFZ-resistant colonies were passed once on CYE agarwithout GFZ, and then on CYE agar containing GFZ 50 μg/ml. Onlyone-GFZ-resistant colony was obtained after the second passage on CYEagar containing GFZ 50 μg/ml. Well over 10¹² colonies were screened forresistance to GFZ.

Effect of GFZ in the growth of the L. pneumophila F4b variant in HL-60cells. Tissue culture medium RPMI 1640 was obtained from JRHBiosciences, Lenexa, Kans.; L-glutamine (LGN) was obtained fromMediatech, Herndon, Va., and phorbol 12-myristate 13-acetate (PMA) wasobtained from Sigma. Normal human serum (Ultraserum™) was obtained fromGemini Bio-Products, Calabasas, Calif. Promyelocytic HL-60 cells weredifferentiated into macrophage-like cells by incubation with 10 ng/mlPMA in RPMI with 2 mM GLN and 10% NHS in Teflon wells at 37° C. for 24hours. These cells were washed, resuspended at 4×10⁶ cells/ml in RPMI-2mM GLN-10% NHS, and mixed with L. pneumophila F4b (4×10⁴ CFU/ml) whichhad been grown for 2 days on ABCYE plates (final multiplicity ofinfection of 0.01). 100 μl aliquots of the suspension were plated ineach well of a 96 well microtiter plate. The plates were centrifuged topellet the cells and bacteria and incubated at 37° C. for 2.5 hours toallow phagocytosis. 100 μl of fresh medium (+/−) 2× the final GFZconcentration, was added to the wells, and the plates were incubated at37° C. At the times indicated, the cells and medium were harvested andassayed for L. pneumophila CFUs as described [58]. Data from theexperiments are expressed as the average (+/−) the S.E.M (n=2).

GFZ Induces the Accumulation of Polyhydroxybutyrate in LegionellaPnuemophila

Gemfibrozil (Lopid™), a drug used to treat hyperlipidemia due to highserum triglycerides, inhibits the growth of pan-sensitive andmultiple-drug resistant Mycobacterium tuberculosis, of Legionellapneumophila, the causative agent of Legionnaire's disease, and ofseveral other bacterial pathogens and yeast. The finding that GFZ(100-200 μg/ml) inhibited four pan-sensitive and twenty two drugresistant strains of M. tuberculosis within a two-fold concentrationrange, suggested that GtZ might have a novel mechanism of action.

The inability to generate GFZ-resistant L. pneumophila mutants by EMSmutagenesis (supra), made it difficult to search for resistancemechanisms and putative targets.

Therefore, the morphology of L. pneumophila following incubation withGFZ was examined by transmission electron microscopy (TEM) to determinewhether GFZ had a visible effect on cell structure that might provide aclue to its mechanism of action.

RESULTS

Analysis of L. pneumophila Incubated on CYE Agar in the Presence orAbsence of GFZ by Transmission Electron Microscopy

By TEM, L. pneumophila grown on CYE agar in the absence of GFZ,contained occasional, small, electron-lucent cytoplasmic inclusionssimilar to those reported by others (FIGS. 15A-D) [94,95]. However, whenL. pneumophila were grown on CYE agar containing 30 μg/ml of GFZ, twopopulations were observed. The bacteria either appeared distended bynumerous, large, electron-lucent, cytoplasmic inclusions, or had amarked absence of electron-lucent inclusions (FIG. 15B). The smallelectron-lucent inclusions found in L. pneumophila grown in the absenceof GFZ are reported to contain polyhydroxybutyrate (PHB), polymers of3-hydroxybutyric acid (3-HB) [96,105].

Although 10 μg/ml of GFZ inhibits growth of 10⁶ L. pneumophila CFUs/mlin AYE broth, and of individual colonies on CYE agar, higherconcentrations of GFZ are required to inhibit growth of a high densityL. pneumophila innoculum (10⁹-10¹⁰ CFUs) on CYE agar. When a largenumber of L. pneumophila is added to CYE plates containing 30 μg/ml ofGFZ, the bacteria grew to form a sparse lawn. This lawn providedsufficient L. pneumophila for analysis. The ability of a large innoculumto grow on CYE medium containing GFZ at 3× the broth MIC is consistentwith reports that an innoculum size affects the apparent MIC of anantibiotic for a given bacteria [76,97]. Additionally, the presence ofcharcoal in CYE plates increases the MIC of many antibiotics.Substitution of potato starch for charcoal generally results in lowerMIC values although this technique was not used here [76].

Nile Blue A Staining of L. pneumophila Grown in the Presence or Absenceof GFZ

Nile Blue A (NBA), a fluorescent dye which stains PHB granules inbacteria [98], was used as a preliminary screen to assess whether thelarge electron-lucent granules seen by electron microscopy, containedPHB. Nile Blue A staining allowed the simultaneous visualization oflarger populations of bacteria. L. pneumophila grown on CYE agar in thepresence or absence of GFZ 30 μg/ml, was harvested and stained with NBA.L. pneumophila grown in the presence of GFZ contained many large,distending, brightly fluorescent NBA-stained inclusions (FIGS. 15A-D),similar to the inclusions seen by EM. L. pneumophila grown in theabsence of GFZ contained fewer, and smaller, NBA-stained inclusions thanL. pneumophila grown in the presence of GFZ (FIGS. 15A-D). Thus,consistent with the EM findings, GFZ treated L. pneumophila containedpopulations of bacteria in which the cytosolic space was filled withNBA-stained material and populations of bacteria containing few if anyNBA-stained inclusions.

Gas Chromatography-mass Spectrometry (GC-MS) of L. pneumophila Grown inthe Presence or Absence of GFZ

Although Nile Blue A is reported to be a specific stain for PHB, asopposed to glycogen, polyphosphates, and other bacterial inclusions, ithad not been shown to be specific for polyhydroxybutyrate versus otherpolyhydroxyalkanoates.

While L. pneumophila is reported to contain PHB granules, some bacteriaare known to respond to alterations in growth conditions by varying thecomposition of their granules, (i.e. polymerization of short chaincarboxylic acid monomers other than 3-HB) [99,100]. Therefore, GC-MS wasused to assess the composition of the inclusions. L. pneumophila grownin the presence or absence of GFZ was subjected to hydrochloric acidpropanolysis [101] to transesterify 3-hydroxyalkanoate monomers withpropanol to form the corresponding propyl esters. Propyl esters wereextracted into dichloroethane and analyzed by GC-MS. A fifty-foldincrease in the amount of 3-HB-propyl ester was observed in L.pneumophila grown for three days on CYE agar in the presence of GFZ,relative to L. pneumophila grown on CYE agar in the absence of GFZ (FIG.16). The difference between the presence and absence of GFZ for PHB wasanalyzed by a student's t-test and found to be significant with t=−6.05,corresponding to a p<0.05 for three independent experiments.

TEM and Nile Blue A staining of L. pneumophila grown in the presence ofa subinhibitory concentration of GFZ indicated that GFZ induced theaccumulation of PHB-containing granules in a majority of the bacteria.However, populations of bacteria lacking significant accumulation of PHBwere also detected. In experiments not reported, log phase L.pneumophila contained a lower percentage of PHB than stationary phasebacteria. Since the viability of the population of GFZ-treated L.pneumophila lacking large PHB was not assessed, it is unclear whetherthese bacteria were growing and “resistant” to the concentration of GFZused, or were dead and therefore unable to form inclusions.

L. pneumophila is not unique in its ability to form granules containingPHB. Several bacterial species are reported to accumulate granulescontaining polyhydroxyalkanoates (PHA), including Alcaligenes eutrophus,Bacillus megatarium, Pseudomonas oleovorans, Pseudomonas aeruginosa, andsome Rhodococcus, Corynebacterium, and Nocardia strains [102,103]. SinceGFZ did not inhibit the growth of Pseudomonas aeruginosa, the ability toform PHA granules is not correlated with GFZ susceptibility.

PHAs are natural polyesters composed of up to 1,000 b-hydroxyacylmonomer units, 3 to 14 carbons in length. The function of these storedPHAs may be to act as an oxidizable substrate during oxygen limitation,as a carbon and energy source, or as a protective mechanism against thedegradation of cellular components such as RNA and protein duringnutrient deprivation [102,103]. Since L. pneumophila are able to utilizeexogenous 3-HB as a carbon source [96], it is likely that their PHBstores contribute to their ability to maintain ATP content and survivein tap water for many months [104,105].

PHAs usually accumulate in bacteria in response to phosphorous, oxygen,nitrogen, or iron limitation in the presence of a carbon source[105,106]. However, to the best of our knowledge, these conditions werenot present in our experiments. We noted however, that PHB synthesis andfatty acid synthesis utilize similar precursors and intermediates.Therefore, we hypothesized that the accumulation of PHB was due to aGFZ-mediated inhibition of fatty acid synthesis (FIG. 17), resulting inthe accumulation of fatty acid precursors and intermediates before theblock, and their subsequent shunting into PHB synthesis (FIG. 18).

Bacteria that synthesize branched chain fatty acids, such as L.pneumophila, can utilize both acetyl CoA and butyryl CoA as fatty acidprecursors [13] [107] [108]. Condensation of two acetyl-CoA molecules isoften the first step in PHB synthesis. Butyryl-CoA has been shown to beincorporated into PHB without degradation to acetyl CoA. Presumably thisoccurs following oxidation of butyryl-CoA to crotonyl-CoA, and hydrationto (R)-3-HB-CoA by enzymes involved in β-oxidation [109]. Additionally,an enzyme PhaG in P. putida can mediate the conversion of 3-HB-ACP, anintermediate in fatty acid synthesis, into 3-HB-CoA, the monomer unitfor PHB synthesis. Experiments described in supra are consistent withthe hypothesis that GFZ causes the accumulation of fatty acidintermediates in L. pneumophila and that these intermediates are likelyto be responsible for the GFZ-induced accumulation of PHB reported inthis study.

Methods and Materials

TEM of L. pneumophila. L. pneumophila was grown for two days on CYEplates, harvested, and resuspended to a concentration of 10¹⁰-10¹¹CFU's/ml in AYE. 10⁹-10¹⁰ bacteria were added to CYE agar medium withoutor with GFZ (30 μg/ml). The plates were incubated for three days at 37°C. The bacteria were harvested, pelleted and fixed by resuspension in2.5% glutaraldehyde for 45 minutes at 25° C., rinsed in 0.1 M NaPO₄buffer (pH_(7.5)) and stored in this buffer overnight, rinsed 2× in 0.1Mcacodylate buffer (pH 7.35), postfixed in 1% osmium tetroxide, rinsed incacodylate buffer pH 7.35, rinsed in 0.1 M NaAc buffer (pH 6.0), stainedin 1% uranyl acetate in 0.1 M NaAc buffer (pH 6.0) for 2.5 hours at 4°C., dehydrated through graded alcohols, embedded in epoxy resin,sectioned into 600 Å sections with a Sorvall MT6000 ultramicrotome, andmounted on 200 mesh copper grids. The sections were stained with uranylacetate for 15 minutes followed by lead citrate for 10 minutes andexamined by TEM using a JEOL1200EX electron microscope.

Nile Blue A Staining of L. pneumophila. L. pneumophila were grown fortwo days on CYE plates, harvested, and resuspended at a concentration of10¹⁰-10¹¹ CFU's/ml in AYE. 10⁹-10¹⁰ a bacteria were added to CYE agarmedium without or with GFZ (30 μg/ml). The plates were incubated forthree days at 37° C. The bacteria were harvested, resuspended in water,smeared on a glass slide and heat fixed. The slides were incubated in a1% aqueous solution of Nile Blue A for 10 minutes at 55° C., washed withtap water, destained for 1 minute in 8% aqueous acetic acid, and airdried. The smears were remoistened with water, covered with a No. 1glass cover slip, and examined by fluorescence microscopy at 460 nm.

Gas chromatography-mass spectrometry. Identification and quantitation of3-hydroxybutyrate (3-HB) was performed by gas chromatography- massspectrometry (GC-MS). 3-HB propyl esters were formed for analysis byhydrochloric acid propanolysis of lyophilized bacteria using benzoicacid as an internal standard [101]. Hydrochloric acid, propanol, benzoicacid, and dichloroethane (DCE) were all obtained from Sigma. In brief,L. pneumophila were grown on CYE agar in the absence or presence of asubinhibitory concentration of GFZ (30 μg/ml) for three days, harvested,and lyophilized. 2 mls DCE, 2 mls acidified propanol, and-200 μl ofinternal standard solution (0.8 mg/ml of benzoic acid in 1-propanol)were added to 40 mg of lyophilized bacteria and the mixture wasincubated for 2 hrs at 100° C. After cooling to room temperature, thesample was washed with 4 mls water. The DCE-propanol phase was reservedand stored at 4° C. A standard curve was constructed by converting knownquantities of 3-HB (Sigma) and benzoic acid to their propyl esters,using hydrochloric acid propanolysis, as before.

Prior to analysis, propyl esters in dichloroethane were dried under astream of nitrogen and resuspended in an equivalent volume of ethylacetate. A 2 μl aliquot of the DCE propanol extract of each sample wasanalyzed after splitless injection into a Hewlett Packard 5987A GC/MSequipped with a DB-1 fused-silica capillary column (30 m×0.2 mm) usinghelium as the carrier gas. The temperatures of the injector and thesource were 220° C. and 200° C., respectively. The column temperatureprogram started at 40° C. for 1 min, and increased at a rate of 8°C./min to 200° C. Samples were ionized by electron impact (70 eV). Theabundance of ions at m/e 105 was used to quantitate benzoic acid, whilethe abundance of ions at m/e 87 was used to quantitate 3-HB.

GFZ Inhibits Lipid Synthesis in Legionella Pneumophila

Previous work demonstrated that growth of several bacterial species andyeast strains was inhibited by GFZ. The observation that M. tuberculosisstrains encoding resistance to many different antibiotics were sensitiveto GFZ, suggested that GFZ inhibited M. tuberculosis, and likely otherbacteria, by a novel mechanism. The observation that GFZ stimulated PHBaccumulation, led to the hypothesis that GFZ inhibited an enzyme inbacterial fatty acid synthesis resulting in the accumulation ofprecursors or intermediates which were shunted into PHB synthesis (FIG.19). Experiments are described herein demonstrating the ability of GFZto inhibit ¹⁴C-acetate incorporation into bacterial fatty acids. Thesefindings confirm that GFZ targets bacterial fatty acid synthesis.

Fatty acids are synthesized by the successive addition of malonyl-ACP toa primer-ACP or a fatty acyl-ACP (FIG. 20). Malonyl-ACP is synthesizedfrom acetyl-CoA which is itself a product of D-oxidation of fatty acids,or, of decarboxylation of pyruvate during growth on sugar. Many bacteriacan also utilize exogenous acetate as a growth substrate. Acetate fromthe medium diffuses into the cytoplasm where it is converted toacetyl-CoA. Acetyl Co-A is subsequently used for oxidation via thetricarboxylic acid (TCA) cycle, for replenishment of intermediates ofthe TCA cycle, for leucine biosynthesis, and for lipid biosynthesis[112]. While L. pneumophila is capable of oxidizing acetate [96,113],¹⁴C-acetate added to L. pneumophila cultures is primarily incorporatedinto the lipid fraction [113]. Therefore, ¹⁴C-acetate is useful as atracer of fatty acid synthesis in this bacterium.

Fatty acid synthesis in most bacteria and plants is carried out bydiscrete, separable enzymes, which are collectively described as a TypeII fatty acid synthetase (Type II FAS) system [5]. In contrast, fattyacid synthesis in mammalian cells is carried out by a homodimer of asingle polypeptide encoding seven distinct enzymatic functions,characteristic of a Type I fatty acid synthetase (Type I FAS) [4].Differences between the human and bacterial fatty acid synthetic enzymesmay account for the ability of GFZ to inhibit fatty acid synthesis incertain bacteria, without affecting the viability of mammalian cells.

RESULTS

¹⁴C-acetate Incorporation into Whole L. pneumophila

To test whether GFZ inhibited fatty acid synthesis, ¹⁴C-acetate wasadded to the medium of log phase L. pneumophila in AYE broth in thepresence of increasing concentrations of GFZ (FIG. 21). Cerulenin, aknown inhibitor of bacterial and mammalian fatty acid synthesis, wasused as a control. Concentrations of GFZ as low as 10 μg/ml (40 μM)inhibited of ¹⁴C-acetate incorporation into wild type L. pneumophilawithin 15 minutes after the addition to the medium. Fifty percentinhibition relative to the control was achieved with a GFZ concentrationof 25 mg/ml (100 mM) within 15 minutes of the drugs addition to themedium. However, inhibition of ¹⁴C-acetate incorporation into F4b, theL. pneumophila derived mutant with moderate resistance to GFZ (MIC₉₉=50μg compared to 10 μg/ml for wild type L. pneumophila) required 100 μg/ml(400 μM) GFZ to inhibit ¹⁴C-acetate incorporation by 50% (FIG. 22).

To confirm that incorporation of ¹⁴C-acetate into TCA precipitablematerial in whole bacteria accurately reflected ¹⁴C-acetate utilizationfor fatty acid synthesis, I analyzed ¹⁴C acetate incorporation intochloroform/methanol extractable material from L. pneumophila grown inthe presence or absence of GFZ. L. pneumophila was incubated for onehour in medium containing ¹⁴C-acetate and increasing concentrations ofGFZ. The bacteria were then pelleted, extracted withchloroform/methanol, and the extracts were analyzed by thin laterchromatography (TLC). Assessment of the amounts of ¹⁴C radiolabelrecovered in the chloroform/methanol extracts and autoradiography of theTLC plates (FIG. 23), confirmed that GFZ inhibited ¹⁴C-acetateincorporation into fatty acids in a dose dependent manner. TLC analysisshowed that the decrease in ¹⁴C-acetate incorporation was equallydistributed among the various fatty acid containing moieties. We drawtwo conclusions from this experiment. First, GFZ inhibits fatty acidsynthesis in L. pneumophila. Second, it does so by blocking an earlystep in fatty acid synthesis, since ¹⁴C-acetate incorporation into allfatty acid containing lipids was inhibited equally.

¹⁴C-Acetate Incorporation into L. pneumophila Lysates

To determine whether GFZ inhibited ¹⁴C-acetate incorporation into lipidsin cell lysates, lysates were prepared from log phase L. pneumophila andincubated for one hour at 37° C. in 50 mM TrisHCl buffer (pH 7.6)containing ATP, Mg⁺⁺, CoA, ¹⁴C-acetate, and GFZ. As observed with intactbacteria, GFZ inhibited ¹⁴C-acetate incorporation into TCA-5precipitable material in these lysates. Further analysis ofchloroform/methanol extracts of these lysates confirmed that the¹⁴C-acetate was largely incorporated into lipids (FIG. 24).

While GFZ inhibited ¹⁴C-acetate incorporation into fatty acids in thelysate, it was not as effective an inhibitor in these lysates as it wasin whole cells. 0.4 mM GFZ only inhibited ¹⁴C-acetate incorporation intoTCA precipitable material in a lysate by 15%, while 0.4 mM GFZ inhibited¹⁴C-acetate incorporation into TCA precipitable material in whole cellsby greater than 90% (FIG. 21). Similarly, cerulenin was a less effectiveinhibitor of ¹⁴C-acetate incorporation into lysates than whole cells(compare FIGS. 21 and 24).

Control experiments showed that lysates that had been boiled prior to¹⁴C-acetate addition, or, had been pre-incubated with 10 mM EDTA, didnot incorporate ¹⁴C-acetate into TCA precipitable material (FIG. 25).EDTA inhibits fatty acid synthesis by chelating Mg⁺⁺ which is a requiredcofactor for ATP-dependent enzymes. In the presence of EDTA, CoAsynthase is unable to form acetyl-CoA so malonyl-ACP is not formed andelongation does not occur.

The effect of a second fibric acid, bezafibrate (BZF) on ¹⁴C-acetateincorporation in L. pneumophila lysates was compared with that of GFZand cerulenin. Surprisingly, BZF was a better inhibitor than GFZ in alysate (FIG. 25).

Effect of GFZ Analogs on ¹⁴C-acetate Incorporation

Structural analogs of GFZ were also tested for inhibition of fatty acidsynthesis in whole cells (FIG. 26). L. pneumophila cultures wereincubated in medium containing ¹⁴C-acetate and each of seven GFZ analogs(FIGS. 27 A-B) (a generous gift from TEVA pharmaceuticals) at a 0.4 mMconcentration. Analogs C and D were found to be better inhibitors at 0.4mM than GFZ at this concentration. Analog B was as effective as GFZ.Dose response experiments were performed for analogs C and D (FIGS.28A-B). Analogs C and D at a concentration of 0.4 mM, inhibited¹⁴C-acetate incorporation into L. pneumophila by 50%. In contrast, aconcentration of 0.1 mM (25 μg/ml) GFZ was required to effect a 50%inhibition of ¹⁴C-acetate incorporation (FIG. 21). Additionalcommercially available compounds with structural similarity to GFZ (FIG.26) were examined at concentrations of 0.5 mM. Of those tested,salicyclic acid, clofibric acid, and P-aminosalicyclic acid demonstratedsome efficacy, although not as great as that found with GFZ at 0.4 mM(FIG. 28 B).

DISCUSSION

The finding that GFZ inhibited ¹⁴C-acetate incorporation into lipids, asmeasured by TCA precipitation and by chloroform/methanol extraction ofL. pneumophila cultures and lysates, was consistent with the hypothesisthat GFZ inhibited fatty acid synthesis. The dose-response studiesdemonstrating that ¹⁴C-acetate incorporation is inversely andproportionately related to GFZ concentration, suggested that GFZ had adirect effect on fatty acid synthesis. The argument that GFZ has adirect effect on fatty acid synthesis is supported by the GFZ-mediatedinhibition of ¹⁴C-acetate incorporation into lysates.

Cerulenin, an inhibitor of the β-ketoacylsynthase reaction in bacteriaand eukaryotes [25,115] with MIC values ranging from 1 to 100 μg/ml}[116], was used as a positive control for the inhibition ¹⁴C-acetateinto fatty acids [117]. The inhibitory action of cerulenin on β-ketoacylsynthase is irreversible; 1 mol of cerulenin binds to 1 mol ofβ-ketoacyl synthase when inhibition approaches 100% [115]. Cerulenin isalso a potent inhibitor of the mammalian fatty acid synthetase enzyme(FAS) [25], which contains a β-ketoacyl synthase domain, and for thisreason is not useful as an antibiotic in humans. However, it is beingpursued as an anticancer drug since certain types of tumors (e.g.ovarian, endometrial, breast, colorectal, and prostate) overexpress FAS[118,119].

Similar to GFZ, cerulenin had an immediate and sustained inhibitoryeffect on the incorporation of ¹⁴C-acetate into L. pneumophila lipids.Cerulenin at 100 μg/ml (0.45 mM) was a slightly better inhibitor of¹⁴C-acetate incorporation than GFZ at 100 μg/ml, 0.4 mM, in whole L.pneumophila. The observation that GFZ is nearly as potent as ceruleninin whole L. pneumophila indicates that it may also have a one to one(drug to target) relationship. The observation that GFZ is not as potentas cerulenin in lysates, indicates that GFZ may need to be metabolizedto an active form.

In our studies, GFZ caused a similar dose-dependent decrease in¹⁴C-acetate incorporation into TCA precipitable material and intochloroform/methanol extractable material. Chloroform/methanol extractionis reported to selectively extract up to 99% of the total lipids from asample [120,121]. Since bacteria do not synthesize sterols, the majorityof the incorporated ¹⁴C-acetate in a chloroform/methanol extract will befound in phospholipids, free fatty acids bound to ACP or CoA, lipid A ofthe lipoploysaccharaide (LPS), the lipoproteins of the outer membrane,and polyhydroxyalkanoate. Other minor lipid species comprise less than1% of total bacterial cell lipids. These include ubiquinone[5,24,122-124] and the lipid containing coenzymes biotin and lipoicacid.

Chloroform/methanol extractions were performed using the method of Blighand Dyer as this method required smaller volumes of chloroform andmethanol, and had been used successfully in L. pneumophila and otherbacteria [41,125]. Although TCA precipitation is not selective forlipids, it is often utilized to assess ¹⁴C-acetate incorporation intobacterial lipids [40,113,126-1281]. Tesh et al [113] examined theincorporation of ¹⁴C-acetate into the TCA soluble and thechloroform/methanol soluble fractions of L. pneumophila over a 12-15hour time period. They demonstrated that 72% of the total radioactivitywas recovered in the chloroform/methanol soluble fraction, while 91% wasrecovered in the TCA precipitate [113].

Acetyl-CoA is utilized by many bacteria as a primer for PHB synthesis.Further, increased pools of acetyl CoA due to inhibition of the TCAcycle in the PHB producing bacteria Azotobacter vinelandii and R.eutropha, resulted in an increased flux of acetyl-CoA into PHB synthesis[129,130]. If L. pneumophila utilizes acetyl-CoA as a precursor for PHBsynthesis, then the greater recovery of radiolabel in the TCAprecipitable fraction may be due to a greater recovery of PHB in the TCAprecipitate.

Due to our use of log phase L. pneumophila, which do not accumulate PHB,and the length of our incubations (i.e. 15-60 minutes of ¹⁴C-acetateexposure), it is unlikely that significant amouts of ¹⁴C-acetate wereincorporated into PHB. However, a small percentage of the ¹⁴C labelrecovered in our TCA precipitable fractions, could be at least partiallydue to the incorporation of ¹⁴C-acetate into PHB. Incorporation of¹⁴C-acetate into PHB would be expected to increase with time asprecursors and intermediates accumulated, and the enzymes responsiblefor synthesizing PHB were induced. An experiment comparing ¹⁴C-acetateincorporation into PHB, in L. pneumophila incubated in the absence andpresence of GFZ over a longer time period would address the hypothesisthat inhibition of fatty acid synthesis by GFZ shunts fatty acidprecursors/intermediates into PHB synthesis.

The lysate experiments were performed to confirm the results obtainedfrom the whole cell experiments in which GFZ inhibited the incorporationof ¹⁴C-acetate into lipids. In addition to confirming these results, thelysate experiments revealed that BZF, a related fibric acid used totreat hypertriglyceridemia, also inhibits lipid synthesis in L.pneumophila. Since BZF does not inhibit growth of intact L. pneumophila,it is likely that BZF does not penetrate the membrane, or, is asubstrate for an efflux transporter in the L. pneumophila membrane.Experiments to assess permeability and active transport usingradioactive BZF might resolve these issues.

As a first step in determining what aspects of the GFZ molecule areimportant for the growth inhibitory effects of GFZ, the ability of otherstructurally similar compounds to inhibit lipid synthesis in whole L.pneumophila was examined. In general, a free carboxylate moiety linkedto a hydrophobic moiety, such as an aliphatic chain, appeared mostimportant. Hydrophobicity is likely to be important for membranepermeability. Substitutions on the aromatic ring that increased thehydrophobicity of TEVA analogs C and D, may have increased the membranepermeability thereby accounting for their increased antibacterialactiviLy against L. pneumophila.

The presence of the carboxylate group is likely to be important for theactivation of GFZ by L. pneumophila. GFZ, like free fatty acids containsa carboxylate group linked to analiphatic chain. Therefore, GFZ, likelong chain fatty acids (e.g. C₁₂-C₁₈ fatty acids), may be converted toits CoA derivative concommitant with transport into bacteria [131-135].Alternatively, GFZ may be converted to its ACP derivative, similar toshort chain fatty acids (e.g. C₆-C₁₁ fatty acids), and a minorproportion, (2%) of long chain fatty acids in E. coli [136]. Analog B,which contains an amide instead of a carboxylate, but is still capableof inhibiting fatty acid synthesis, may have a different mechanism ofinhibiting fatty acid synthesis. Alternatively, it may be converted to acarboxylate by L. pneumophila. Experiments to test this possibility mayyield insights into the mechanisms by which TEVA analog B acts to block¹⁴C-acetate incorporation into lipids in L. pneumophila.

In conclusion, GFZ was found to be an inhibitor of lipid synthesis in L.pneumophila. This finding was consistent with reports that GFZ and otherfibrates inhibit fatty acid elongation in mammalian systems [67,68].

Materials and Methods

¹⁴C-acetate incorporation into whole L. pneumophila. L. pneumophila weregrown to log phase (OD=0.6-1.0) in AYE broth at 37° C. 0.5-1.0 mlaliquots were pelleted and resuspended in an equivalent volume of freshAYE medium. GFZ and other inhibitors were added to the cultures.¹⁴C-acetate (specific activity 48.9 mCi/mmol) (Sigma) was addedimmediately thereafter (final concentration 5 μCi/ml) and the cultureswere incubated at 37° C. At various time points, 100 μl aliquots of theculture were added to 600 μl 12% TCA on ice; final concentration 10%TCA. TCA precipitates were pipetted onto a Whatman GF/F glass microfibrefilter and washed with 30 ml of cold 10% TCA. The filters were dried andradioactivity was assessed by scintillation counting[40,113126,127,139]. Alternatively, at various time points the bacteriawere placed on ice, pelleted at 4° C., washed three times with 1 ml ofice cold AYE, and the pellet was extracted with 400 μl ofchloroform/methanol (1:1 vol:vol). The extract was washed with 180 ml ofwater according to the method of Bligh and Dyer [113,120]. 5 μl of thechloroform layer was combined with 5 mls of CytoScint and counted in ascintillation counter. GFZ, salicylate, para-aminosalicylate, isoniazid,clofibrate, 4-hydroxypropionate were obatined from Sigma;3,4-hydroxypropionate was obtained from Aldrich.

Thin layer chromatography. 50 μl of each chloroform/methanol extract wasspotted onto EM Science™ Silica Gel 60 F254 plates (Fisher) anddeveloped in chloroform:methanol:acetic acid (65:25:2) until the solventfront was halfway up the plate. The TLC plate was dried, and thendeveloped in chloroform:methanol:sodium acetate pH 3.4 until the solventfrom was 1 cm from the top of the plates. The plates were air dried,exposed to iodine vapors to visualize lipid bands. The presence of¹⁴C-acetate labeled lipids was assessed by exposing the TLC plate toFuji™ RX film for five days at −80° C.

¹⁴C-acetate incorporation into lysates. One liter of log phase L.pneumophila, grown in AYE broth at 37° C., was pelleted, frozen, thawed,resuspended in 5 mls lysis buffer (50 mM Tris HCl pH 7.6, lysozyme 1mg/ml, EDTA 1 mM, and one Complete™ protease inhibitor tablet) andsonicated at 4° C. The lysate was centrifuged for 10 minutes at 8,000×gat 4° C. to remove intact cells. 50 ul of lysate were combined with 50ul of a cocktail consisting of 50 mM Tris HCl (pH 7.6), 5 mM MgCl₂, 5 mMATP, 1 MM CoA, 1 mM DTT, 1 mM NADH, 1 mM NADPH, and ¹⁴C-acetate (10μCi/ml) in 1.5 ml eppendorf tubes on ice. Boiled lysates were preparedby heating a 50 μl aliquot of the lysate for 10 minutes in a boilingwater bath prior to the addition of the above cocktail. GFZ, BZF, CER,were added to the lysaztes to a final concentration of 2 mM. EDTA wasadded to the lysates to a final concentration of 10 mM. The reactionmixtures were incubated for 10-30 minutes at 37° C. At the timesindicated, the 100 μl reaction were placed on ice and precipitated bythe addition of 600 μl of ice cold 12% TCA. The precipitates werepelleted in a microfuge at 4° C., and washed 3× with 1 ml of 10% TCA ineach wash. The washed TCA precipitated material was extracted with 200μl of chloroform/methanol (1:1), and the extract was washed with 100 ulof water. The aqueous layer was removed, washed with 100 μl ofchloroform/methanol (1:1), and the two organic extracts were combined.The entire organic extract was added to a scintillation tube, evaporatedovernight and counted in 5 mls of CytoScint with a scintillationcounter.

GFZ Inhibits an Enoyl Reductase from Legionella pneumophila

The finding that L. pneumophila. accumulated polyhydroxybutyrate (PHB)in response to GFZ led us to hypothesize that GFZ inhibited enzyme(s)involved in fatty acid synthesis/elongation. Support for this hypothesiswas obtained by studies of the ability of GFZ to inhibit ¹⁴C-acetateincorporation into L. pneumophila lipids. Since GFZ inhibited lipidsynthesis in L. pneumophila, we reasoned that the most likely targetwould be a regulatory enzyme in fatty acid synthesis.

Putative targets included the β-ketoacylsynthase, the target of thefatty acid synthesis inhibitors cerulenin [115] and thiolactomycin [28];b-hydroxydecenoyl dehydrase, the target for the unsaturated fatty acidsynthesis inhibitor N-decenoyl-N-acetylcysteamine [33]; and enoylreductase, the target for diazoborines [140], triclosan [40], andisoniazid [1] (FIG. 29).

Since the FabI enoyl reductase of E. coli was reported to be anessential enzyme involved in regulating the rate of fatty acidsynthesis, we chose to examine this protein for sensitivity to GFZ. Asecond reason for the selection of enoyl reductase, was the observationthat inhibition of the FabI enoyl reductase in E. coli stimulated theaccumulation of 3-hydroxybutyryl-ACP (3-HB-ACP) [17]. Under normalconditions, crotonoyl-ACP, the substrate for E. coli FabI, is unstableand is present at {fraction (1/10)} the level of 3-HB-ACP [14].Therefore, inhibition of FabI in E. coli stimulates the accumulation of3-HB-ACP rather than crotonoyl-ACP. If L. pneumophila contains a PhaGhomolog capable of converting 3-HB-ACP to 3-HB-CoA, then GFZ-mediatedenoyl reductase inhibition might result in the accumulation of aprecursor for PHB synthesis.

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1. A method of selecting a compound which inhibits the enzymaticactivity of enoyl reductase which comprises; (A) contacting enoylreductase with the compound; and (B) measuring the enzymatic activity ofthe enoyl reductase of step (A) compared with the enzymatic activity ofenoyl reductase in the absence of the compound, and selecting thecompound which inhibits the enzymatic activity of enoyl reductase,wherein the compound has the structure:

or a pharmaceutically acceptable salt ester thereof, wherein (i) each ofR₁, R₂, R₃, R₄, R₅, and R₆ is independently selected from the groupconsisting of —H, —F, —Cl, —Br, —I, —OH, —OR₇, —CN, —COR₇, —SR₇,—N(R₇)₂, —NO₂, —(CH₂)—OR₇, —COSR₇, —C(═O)—OH, —CONH₂, —NH₂, a straightchain or branched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl,thicalkyl, methylene thioalkyl, acyl, phenyl, substituted phenyl andheteroaryl; (ii) the compound is in the form of acyl carrier proteinmetabolite; (iii) L is —N—, —S—, —O—, —C≡C— or —CH₂—; (iv) R₇ isindependently selected from the group consisting of —H, —F, —Cl, —Br,—I, —OH, —CN, —COH, —SH, —NH₂, —NHCOH, —(CH₂)OH, a straight chain orbranched, substituted or unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl, thioalkyl,methylene thioalkyl, acyl, phenyl, substituted phenyl and heteroaryl;(v) A is selected from the group consisting of —N₂—, —NH—, —CH═C═CH—,—C≡C—CH(OH)—, C≡C—CH₂—, —CH₂—CH₂—O—, —CH₂—CH₂—CH₂—O—, —S—, —S(═O)₂—,—C(═O)—, —C(═O)—O—, —NH—C(═O)— and —C(═O)—NH—; (vi) Q is independentlyan integer from 1 to 10, and if Q is 1, A may be a (C₁-C₁₀)-alkyl chain,(C₂-C₁₀)-alkenyl chain or (C₂-C₁₀)-alkynyl chain which is branched orunbranched, substituted or unsubstituted and is optionally inturrupted 1to 3 times by —O— or —S— or —N—; and (vii) X is —C(═O)O—, —CH═CH—,phenyl, substituted phenyl, heteroaryl, —O-phenyl (CH₃)₂ —,C(CH₃)₂—C(═O)—NH— or —C(CH₃)₂—C(═O)—O—.
 2. The method of claim 1,wherein the compound contacts enoyl reductase at the site at whichgemfibrozil contacts enoyl reductase.